Polymerase compositions and methods

ABSTRACT

Disclosed herein are modified polymerase compositions exhibiting altered polymerase activity, which can be useful in a variety of biological applications. Also disclosed herein are methods of making and using such compositions. In some embodiments, the compositions exhibit altered properties that can enhance their utility in a variety of biological applications. Such altered properties, can include, for example, altered nucleotide binding affinities, altered nucleotide incorporation kinetics, altered photostability and/or altered nanoparticle tolerance, as well as a range of other properties as disclosed herein.

RELATED APPLICATIONS

This application is a divisional of U.S. Nonprovisional application Ser.No. 15/169,854 filed on Jun. 1, 2016, now allowed, which is a divisionalof U.S. Nonprovisional application Ser. No. 14/710,160, filed May 12,2015, now U.S. Pat. No. 9,365,839, which is a continuation of U.S.Nonprovisional application Ser. No. 13/540,935, filed Jul. 3, 2012, nowabandoned, which is a continuation of U.S. Nonprovisional applicationSer. No. 12/748,359, filed Mar. 26, 2010, now abandoned, which claimsthe filing date benefit of U.S. Provisional Application Nos.:61/164,324, filed on Mar. 27, 2009; 61/184,770, filed on Jun. 5, 2009;61/242,771, filed on Sep. 15, 2009; 61/245,457, filed on Sep. 24, 2009;61/263,974, filed on Nov. 24, 2009; 61/289,388; filed on Dec. 22, 2009;61/293,618, filed on Jan. 8, 2010; 61/293,616, filed on Jan. 8, 2010;61/299,919, filed on Jan. 29, 2010; 61/299,917, filed on Jan. 29, 2010;61/307,356, filed on Feb. 23, 2010. The contents of each of theforegoing patent applications are incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 21, 2015, isnamed LT00052CON_SL.txt and is 78,446 bytes in size.

FIELD

The disclosure relates generally to polymerase compositions and methods.More particularly, the disclosure relates to modified polymerases andtheir use in biological applications including, for example, nucleotideincorporation, primer extension and single molecule sequencingreactions.

BACKGROUND

The polymerases typically catalyze nucleic acid synthesis against anexisting polynucleotide template using Watson-Crick base pairinginteractions, and are useful in a variety of biological applications.Such applications frequently involve the use of labels to visualize oneor more components or products of the polymerase reaction. For example,“sequencing by synthesis” applications typically involve the monitoringof polymerase activity in real time by detecting signals emitted bylabels associated with one or more components of the polymerasereaction. However, many labels cannot be employed in such assays becausetheir presence inhibits polymerase activity. For example, althoughnanoparticles can exhibit superior quantum yield, size tunability,brightness and resistance to photobleaching compared to conventionalorganic, e.g., dye, labels, their utility in polymerase-based assays ishampered by the sensitivity of many polymerases to the presence ofnanoparticles. Furthermore, many polymerases can also exhibit loss ofpolymerase activity upon exposure to excitation radiation, thushampering their use in assays involving labels that require excitationto be detectable. This problem can be exacerbated in the presence ofnanoparticles, resulting in further loss of polymerase activity.

Yet another set of problems revolves around the kinetic behavior of thepolymerase towards nucleotide substrates. Analysis of polymeraseactivity can be complicated by undesirable behavior such as, forexample, the tendency of a given polymerase to dissociate from thetemplate; to bind and/or incorporate the incorrect, e.g., nonWatson-Crick base-paired, nucleotide; or to release the correct, e.g.,Watson-Crick based paired, nucleotide without incorporation. Inaddition, some applications may require the use of polymerasesexhibiting increased residence times or branching ratios for particularnucleotides, so as to increase the duration during which labelednucleotide incorporation can be detected. Finally, although manybiological applications require the use of labeled nucleotides, manypolymerases do not incorporate such nucleotides efficiently, thuslimiting the utility of such polymerase-nucleotide combinations in theseapplications. These and other desirable properties can be enhanced viasuitable selection, engineering and/or modification of a polymerase ofchoice.

It is therefore desirable to develop polymerases having increasedtolerance for the presence of both organic (e.g., conventional dye) andinorganic (e.g., nanoparticle-based) labels, as well as polymerases thatretain higher levels of polymerase activity following exposure toexcitation radiation. There is also a need for polymerases that exhibitimproved reaction kinetics with a particular set of labeled nucleotides.Accordingly, there remains a need in the art for improved polymerasecompositions, and methods of use thereof, which can permitpolynucleotide synthesis with higher efficiency while allowing the useof an expanded repertoire of excitation and/or labeling strategies.

SUMMARY

Provided herein are novel polymerase compositions, methods of makingsuch compositions and methods of using such compositions in variousbiological applications.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a photostability that is at least about 80% understandard photostability assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard photostability assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a nanoparticle tolerance that is at least about 80%under standard nanoparticle tolerance assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard nanoparticle tolerance assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard nanoparticle tolerance assay conditions.

In some embodiments, the disclosure relates to a nucleic acid moleculeencoding any one, some or all of the modified DNA polymerases of thepresent disclosure. The nucleic acid molecule can optionally be DNA orRNA.

In some embodiments, the disclosure relates to a vector comprising a DNAencoding any one, some or all of the modified DNA polymerases asprovided herein.

In some embodiments, the disclosure relates to an isolated host cellcomprising a vector including a DNA encoding any one, some or all of themodified DNA polymerases of the present disclosure.

In some embodiments, the disclosure relates to a method for obtainingthe modified DNA polymerases of the present disclosure. Optionally, themethod comprises purifying the modified DNA polymerase from an isolatedhost cell comprising a vector including a DNA encoding a modified DNApolymerase of the present disclosure.

In some embodiments, the disclosure relates to a method for performing aprimer extension reaction, comprising: contacting a modified DNApolymerase as provided herein with a nucleic acid molecule and anucleotide under conditions where the nucleotide is incorporated intothe nucleic acid molecule by the modified DNA polymerase. Optionally,the nucleotide is a labeled nucleotide, and the label of the nucleotideemits a signal during incorporation of the at least one nucleotide.Optionally, the method further comprises detecting the signal emitted bythe nucleotide label. Optionally, the method further comprises analyzingthe detected signal to determine the identity of the incorporatednucleotide.

In some embodiments, the disclosure relates to a modified DNA polymerasehaving an increased branching ratio in the presence of labelednucleotides relative to a DNA polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified DNA polymerasecomprising an amino acid sequence that is at least about 95% identicalto the amino acid sequence of SEQ ID NO: 7. Optionally, the modified DNApolymerase further includes the amino acid mutation H370R.

DETAILED DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the disclosure by wayof illustrating non-limiting embodiments and examples. The disclosuremay be better understood by reference to one or more of these figures incombination with the detailed description of specific embodimentspresented herein.

FIG. 1 depicts one exemplary theoretical model of the various stages inbinding of a polymerase to a nucleic acid molecule and a nucleotidesubstrate, followed by dissociation of the nucleotide substrate inunaltered state (i.e., a non-productive event) or by incorporation ofthe nucleotide into the nucleic acid molecule.

FIG. 2 depicts an expression vector, pTTQ-B104-exo minus, comprising theamino acid sequence of SEQ ID NO: 8 fused downstream of an isopropylβ-D-1-thiogalactopyranoside (IPTG) promoter in the expression vectorpTTQ.

FIG. 3 depicts the results of an assay measuring the fractionalextension activity, i.e., the fraction of nucleic acid templates thatare extended by at least one nucleotide in a polymerase reaction, of anexemplary modified polymerase comprising the amino acid sequence of SEQID NO: 8, and two exemplary reference polymerases comprising the aminoacid sequences of SEQ ID NO: 15 and SEQ ID NO: 20, respectively.

FIG. 4 depicts the results of an assay measuring the exonucleaseactivity of T7 DNA polymerase, an exemplary reference polymerase, and ofan exemplary modified variant comprising the amino acid sequence of SEQID NO: 7.

FIG. 5 depicts the results of an assay measuring the nanoparticletolerance of an exemplary modified polymerase comprising the amino acidsequence of SEQ ID NO: 19, and an exemplary reference polymerasecomprising the amino acid sequence of SEQ ID NO: 20.

FIG. 6A depicts the results of an assay measuring the photostability ofan exemplary modified polymerase comprising the amino acid sequence ofSEQ ID NO: 19, and an exemplary reference polymerase comprising theamino acid sequence of SEQ ID NO: 20. FIG. 6A shows a polyacrylamide gelloaded with primer extension reactions of B104 and phi29 polymerasesexposed to excitation radiation. FIG. 6B also depicts the results of anassay measuring the photostability of an exemplary modified polymerasecomprising the amino acid sequence of SEQ ID NO: 19, and an exemplaryreference polymerase comprising the amino acid sequence of SEQ ID NO:20. FIG. 6B shows a graph of the primer extension reactions of B104 andphi29 polymerases exposed to excitation radiation.

FIG. 7A depicts time traces of fluorescence at 550 nm from threedifferent stopped-flow assay systems, each comprising a mutant Phi-29polymerase. FIG. 7A shows a time trace of a mutant Phi-29 polymerase andterminally labeled nucleotide triphosphates. FIG. 7B depicts time tracesof fluorescence at 550 nm from three different stopped-flow assaysystems, each comprising a mutant Phi-29 polymerase. FIG. 7B shows atime trace of a mutant Phi-29 polymers and a terminally labelednucleotide tetraphosphates. FIG. 7C depicts time traces of fluorescenceat 550 nm from three different stopped-flow assay systems, eachcomprising a mutant Phi-29 polymerase. FIG. 7C shows a time trace of amutant Phi-29 polymerase and a terminally labeled nucleotidehexaphosphates.

FIG. 8A depicts time traces of fluorescence at 550 nm from two differentstopped-flow assay systems, each comprising different mutant Phi-29polymerase. FIG. 8A shows a time trace of a mutant Phi-29 polymerase andterminally labeled nucleotide hexaphosphates. FIG. 8B depicts timetraces of fluorescence at 550 nm from two different stopped-flow assaysystems, each comprising different mutant Phi-29 polymerase. FIG. 8Bshows a time trace of a mutant Phi-29 polymerase and terminally labelednucleotide hexaphosphates.

FIG. 9 provides a graphical depiction of the various reaction componentsincluded in the real-time single molecule sequencing reaction of Example10.

FIG. 10 depicts exemplary portions of fluorescence time traces detectedat 550 and 647 nm from a single-molecule real time sequencing reactioncomprising a mutant Phi-29 polymerase and terminally-labeled nucleotidehexaphosphates.

FIG. 11 depicts the general structure of a dye-labeled nucleotidehexaphosphate that can be used in conjunction with the labeledpolymerase conjugates disclosed herein.

DETAILED DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO: 1 comprises the amino acid sequence of an exemplarypolymerase of the bacteriophage Phi-29.

SEQ ID NO: 2 comprises the amino acid sequence of a consensus motifnamed Motif 1, also known as Motif A, which can be found within variouspolymerases including polymerases isolated from bacteriophages of thefamily of Phi-29-like bacteriophages.

SEQ ID NO: 3 comprises the amino acid sequence of a consensus motifnamed Motif 2a, also known as Motif B, which can be found within variouspolymerases including polymerases isolated from bacteriophages of thefamily of Phi-29-like bacteriophages.

SEQ ID NO: 4 comprises the amino acid sequence of a consensus motifnamed Motif 3, also known as Motif C, which can be found within variouspolymerases including polymerases isolated from bacteriophages of thefamily of Phi-29-like bacteriophages.

SEQ ID NO: 5 comprises the amino acid sequence of a consensus motifnamed Motif 4, we can be found within various polymerases includingpolymerases isolated from bacteriophages of the family of Phi-29-likebacteriophages.

SEQ ID NO: 6 comprises the amino acid sequence of an exemplarypolymerase of the bacteriophage B103, a member of the Phi-29-like familyof bacteriophages

SEQ ID NO: 7 comprises the amino acid sequence of an exemplary modifiedpolymerase according to the present disclosure.

SEQ ID NO: 8 comprises the amino acid sequence of another exemplarymodified polymerase according to the present disclosure.

SEQ ID NO: 9 comprises the nucleotide sequence of an exemplarypolynucleotide encoding the modified polymerase comprising the aminoacid sequence of SEQ ID NO: 7.

SEQ ID NO: 10 comprises the nucleotide sequence of a second exemplarypolynucleotide encoding the modified polymerase comprising the aminoacid sequence of SEQ ID NO: 7.

SEQ ID NO: 11 comprises the nucleotide sequence of an exemplarypolynucleotide encoding the modified polymerase comprising the aminoacid sequence of SEQ ID NO: 8.

SEQ ID NO: 12 comprises the nucleotide sequence of a second exemplarypolynucleotide encoding the modified polymerase comprising the aminoacid sequence of SEQ ID NO: 8.

SEQ ID NO: 13 comprises the amino acid sequence of a polymerase of thebacteriophage M2Y, a member of the Phi-29-like family of bacteriophages.

SEQ ID NO: 14 comprises the amino acid sequence of a polymerase of thebacteriophage Nf, a member of the Phi-29-like family of bacteriophages.

SEQ ID NO: 15 comprises the amino acid sequence of a polymerase of RB69.

SEQ ID NO: 16 comprises the amino acid sequence of an exemplary peptidelinker, named “H-linker”, useful in linking tags or labels to a proteinsequence of interest.

SEQ ID NO: 17 comprises the amino acid sequence of another exemplarypeptide linker, named “F-linker”, useful in linking tags or labels to aprotein sequence of interest.

SEQ ID NO: 18 comprises the amino acid sequence of an exemplaryHis-tagged version of a protein comprising the amino acid sequence ofSEQ ID NO: 7.

SEQ ID NO: 19 comprises the amino acid sequence of another exemplaryHis-tagged version of a protein comprising the amino acid sequence ofSEQ ID NO: 7.

SEQ ID NO: 20 comprises the amino acid sequence of HP1, a Phi-29polymerase peptide that lacks exonuclease activity and comprises anN-terminal His-tag, an intervening linker sequence, and the D12A andD66A mutations.

SEQ ID NO: 21 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used to construct a primer:template duplex foruse in assays for fractional extension activity as disclosed, forexample, in Example 3.

SEQ ID NOS: 22-25 each comprises the nucleotide sequence of an exemplarypolynucleotide template used to construct a primer:template duplex foruse in assays for fractional extension activity as disclosed, forexample, in Example 3.

SEQ ID NO: 26 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used to construct a primer:template duplex foruse in nucleotide incorporation assays to evaluate polymerase reactionkinetics as disclosed, for example, in Example 7.

SEQ ID NOS: 27-30 each comprises the nucleotide sequence of an exemplarypolynucleotide template used to construct a primer:template duplex foruse in nucleotide incorporation assays as disclosed, for example, inExample 7.

SEQ ID NO: 31 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used to construct a primer:template duplex foruse in nucleotide incorporation assays to evaluate polymerase reactionkinetics as disclosed, for example, in Example 8.

SEQ ID NOS: 32-35 each comprise the nucleotide sequence of an exemplarypolynucleotide template used to construct a primer:template duplex foruse in nucleotide incorporation assays as disclosed, for example, inExample 8.

SEQ ID NO: 36 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used to construct a primer-template duplex foruse in a real time single molecule sequencing assay, as disclosed, forexample, in Example 10.

SEQ ID NO: 37 comprises the nucleotide sequence of an exemplarypolynucleotide template used to construct a primer-template duplex foruse in a real time single molecule sequencing assay, as disclosed, forexample, in Example 10.

SEQ ID NO: 38 comprises the nucleotide sequence of an exemplary hairpintemplate used for three-color nucleotide incorporation reaction asdescribed, for example, in Example 11.

SEQ ID NO: 39 comprises the nucleotide sequence of an exemplarypolynucleotide template used for a four-color nucleotide incorporationreaction as described, for example, in Example 11.

SEQ ID NO: 40 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used for a four-color nucleotide incorporationreaction as described, for example, in Example 11.

SEQ ID NO: 41 comprises the predicted nucleotide sequence that will besynthesized in a four-color nucleotide incorporation reaction using theprimer of SEQ ID NO: 40 in conjunction with the template of SEQ ID NO:39 as described, for example, in Example 11.

SEQ ID NO: 42 comprises the amino acid sequence of a modified B103polymerase comprising the amino acid sequence of SEQ ID NO: 8 andfurther including the mutation H370R as well as a biotinylation site andHis tag fused to the N-terminus of the polymerase.

SEQ ID NO: 43 comprises the nucleotide sequence of a fluorescein-labeledoligonucleotide primer used to measure primer extension activity of apolymerase sample according to an exemplary assay, as described inExample 13.

SEQ ID NO: 44 comprises the nucleotide sequence of an exemplarypolynucleotide template used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 12.

SEQ ID NO: 45 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 12.

SEQ ID NO: 46 comprises the nucleotide sequence of an exemplarypolynucleotide template used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 12.

SEQ ID NO: 47 comprises the nucleotide sequence of an exemplaryoligonucleotide primer used in a stopped-flow assay for nucleotideincorporation kinetics as described, for example, in Example 12.

DETAILED DESCRIPTION

The present disclosure provides for compositions, methods and systemsrelating to modified polymerases and their use in various biologicalapplications. More particularly, provided herein are novel polymeraseshaving altered polymerase activity, label tolerance, photostabilityand/or nucleotide incorporation kinetics as compared to their unmodifiedcounterparts, which can be useful in a wide variety of biologicalapplications.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is set forthherein that is contrary to or otherwise inconsistent with any definitionset forth in the patents, patent applications, published applications,and other publications that are herein incorporated by reference, thedefinition and/or description set forth herein prevails over thedefinition that is incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A LaboratoryManual, Third Edition; Ausubel, F. M., et al., eds., 2002, ShortProtocols In Molecular Biology, Fifth Edition.

Unless otherwise indicated, the numbering of any amino acid residuesdescribed herein will be relative to the sequence of a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6. However, the skilledartisan will appreciate that the actual position within a modifiedpolymerase according to the present disclosure may vary. For example,the modified polymerase may comprise one or more deletions or additionswithin its sequence, thereby altering the numbering of the correspondingamino acid residue in the modified polymerase relative to the B103polymerase having SEQ ID NO: 6.

Throughout this disclosure, various amino acid mutations, including, forexample, amino acid substitutions are referenced using the amino acidsingle letter code, and indicating the position of the residue within areference amino acid sequence. In the case of amino acid substitutions,the identity of the substituent is also indicated using the amino acidsingle letter code. For example, the amino acid substitution F383L,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6, indicates an amino acid substitution wherein a leucine (L)residue is substituted for the normally occurring phenylalanine (F)residue at amino acid position 383 of the amino acid sequence of SEQ IDNO: 6.

As used herein, the terms “link”, “linked”, “linkage” and variantsthereof comprise any type of fusion, bond, adherence or association thatis of sufficient stability to withstand use in the particular biologicalapplication of interest. Such linkage can comprise, for example,covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, oraffinity bonding, bonds or associations involving van der Waals forces,mechanical bonding, and the like. Optionally, such linkage can occurbetween a combination of different molecules, including but not limitedto: between a nanoparticle and a protein; between a protein and a label;between a linker and a functionalized nanoparticle; between a linker anda protein; and the like. Some examples of linkages can be found, forexample, in Hermanson, G., Bioconjugate Techniques, Second Edition(2008); Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniquesfor the Biomedical Sciences, London: Macmillan (1998); Aslam, M., Dent,A., Bioconjugation: Protein Coupling Techniques for the BiomedicalSciences, London: Macmillan (1998).

As used herein, the term “linker” and its variants comprises anycomposition, including any molecular complex or molecular assembly, thatserves to link two or more compounds.

As used herein, the term “polymerase” and its variants comprise anyenzyme that can catalyze the polymerization of nucleotides (includinganalogs thereof) into a nucleic acid strand. Typically but notnecessarily such nucleotide polymerization can occur in atemplate-dependent fashion. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. Some exemplary polymerases includewithout limitation DNA polymerases (such as for example Phi-29 DNApolymerase, reverse transcriptases and E. coli DNA polymerase) and RNApolymerases. The term “polymerase” and its variants, as used herein,also refers to fusion proteins comprising at least two portions linkedto each other, where the first portion comprises a peptide that cancatalyze the polymerization of nucleotides into a nucleic acid strandand is linked to a second portion that comprises a second polypeptide,such as, for example, a reporter enzyme or a processivity-enhancingdomain. One exemplary embodiment of such a polymerase is Phusion® DNApolymerase (New England Biolabs), which comprises a Pyrococcus-likepolymerase fused to a processivity-enhancing domain as described, forexample, in U.S. Pat. No. 6,627,424.

As used herein, the term “polymerase activity” and its variants, whenused in reference to a given polymerase, comprises any in vivo or invitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing the polymerization of nucleotides into a nucleicacid strand, e.g., primer extension activity, and the like. Typically,but not necessarily such nucleotide polymerization occurs in atemplate-dependent fashion. In addition to such polymerase activity, thepolymerase can typically possess other enzymatic activities, forexample, 3′ to 5′ exonuclease activity.

As used herein, the term “nucleotide” and its variants comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase; occasionallyhowever the nucleotide may dissociate from the polymerase withoutbecoming incorporated into the nucleic acid strand, an event referred toherein as a “non-productive” event. Such nucleotides include not onlynaturally occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties. In some embodiments, the nucleotide can optionally include achain of phosphorus atoms comprising three, four, five, six, seven,eight, nine, ten or more phosphorus atoms. In some embodiments, thephosphorus chain can be attached to any carbon of a sugar ring, such asthe 5′ carbon. The phosphorus chain can be linked to the sugar with anintervening O or S. In one embodiment, one or more phosphorus atoms inthe chain can be part of a phosphate group having P and O. In anotherembodiment, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where Rcan be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorusatoms in the chain can have side groups having O, BH₃, or S. In thephosphorus chain, a phosphorus atom with a side group other than O canbe a substituted phosphate group. In the phosphorus chain, phosphorusatoms with an intervening atom other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotidecomprises a label and referred to herein as a “labeled nucleotide”; thelabel of the labeled nucleotide is referred to herein as a “nucleotidelabel”. In some embodiments, the label can be in the form of afluorescent dye attached to the terminal phosphate group, i.e., thephosphate group most distal from the sugar. Some examples of nucleotidesthat can be used in the disclosed methods and compositions include, butare not limited to, ribonucleotides, deoxyribonucleotides, modifiedribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, modified peptide nucleotides,metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

As used herein, the term “nucleotide incorporation” and its variantscomprises polymerization of one or more nucleotides into a nucleic acidstrand.

As used herein, the term “biological activity” and its variants, whenused in reference to a biomolecule (such as, for example, an enzyme)refers to any in vivo or in vitro activity that is characteristic of thebiomolecule itself, including the interaction of the biomolecule withone or more targets. For example, biological activity can optionallyinclude the selective binding of an antibody to an antigen, theenzymatic activity of an enzyme, and the like. Such activity can alsoinclude, without limitation, binding, fusion, bond formation,association, approach, catalysis or chemical reaction, optionally withanother biomolecule or with a target molecule.

As used herein, the term “biologically active fragment” and its variantsrefers to any fragment, derivative or analog of a biomolecule thatpossesses an in vivo or in vitro activity that is characteristic of thebiomolecule itself. For example, the biomolecule can be an antibody thatis characterized by antigen-binding activity, or an enzyme characterizedby the ability to catalyze a particular biochemical reaction, etc.Biologically active fragments can optionally exist in vivo, such as, forexample, fragments which arise from post transcriptional processing orwhich arise from translation of alternatively spliced RNAs, oralternatively can be created through engineering, bulk synthesis, orother suitable manipulation. Biologically active fragments includefragments expressed in native or endogenous cells as well as those madein expression systems such as, for example, in bacterial, yeast, insector mammalian cells. Because biomolecules often exhibit a range ofphysiological properties and because such properties can be attributableto different portions of the biomolecule, a useful biologically activefragment can be a fragment of a biomolecule that exhibits a biologicalactivity in any biological assay. In some embodiments, the fragment oranalog possesses 10%, 40%, 60%, 70%, 80% or 90% or greater of theactivity of the biomolecule in any in vivo or in vitro assay ofinterest.

The term “modification” or “modified” and their variants, as used hereinwith reference to a protein, comprise any change in the structural,biological and/or chemical properties of the protein, particularly achange in the amino acid sequence of the protein. In some embodiments,the modification can comprise one or more amino acid mutations,including without limitation amino acid additions, deletions andsubstitutions (including both conservative and non-conservativesubstitutions).

As used herein, the terms “identical” or “percent identity,” and theirvariants, when used in the context of two or more nucleic acid orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using any one or more of the followingsequence comparison algorithms: Needleman-Wunsch (see, e.g., Needleman,Saul B.; and Wunsch, Christian D. (1970). “A general method applicableto the search for similarities in the amino acid sequence of twoproteins” Journal of Molecular Biology 48 (3):443-53); Smith-Waterman(see, e.g., Smith, Temple F.; and Waterman, Michael S., “Identificationof Common Molecular Subsequences” (1981) Journal of Molecular Biology147:195-197); or BLAST (Basic Local Alignment Search Tool; see, e.g.,Altschul S F, Gish W, Miller W, Myers E W, Lipman D J, “Basic localalignment search tool” (1990) J Mol Biol 215 (3):403-410).

The terms “resonance energy transfer” and “RET” and their variants, asused herein, refer to a radiationless transmission of excitation energyfrom a first moiety, termed a donor moiety, to a second moiety termed anacceptor moiety. One type of RET includes Forster Resonance EnergyTransfer (FRET), in which a fluorophore (the donor) in an excited statetransfers its energy to a proximal molecule (the acceptor) bynonradiative dipole-dipole interaction. See, e.g., Forster, T.“Intermolecular Energy Migration and Fluorescence”, Ann. Phys 2:55-75,1948; Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 2nd ed.Plenum, N.Y. 367-394, 1999. RET also comprises luminescence resonanceenergy transfer, bioluminescence resonance energy transfer,chemiluminescence resonance energy transfer, and similar types of energytransfer not strictly following the Forster's theory, such asnonoverlapping energy transfer occurring when nonoverlapping acceptorsare utilized. See, for example, Anal. Chem. 2005, 77:1483-1487.

The term “conservative” and its variants, as used herein with referenceto any change in amino acid sequence, refers to an amino acid mutationwherein one or more amino acids is substituted by another amino acidhaving highly similar properties. For example, one or more amino acidscomprising nonpolar or aliphatic side chains (for example, glycine,alanine, valine, leucine, isoleucine or proline) can be substituted foreach other. Similarly, one or more amino acids comprising polar,uncharged side chains (for example, serine, threonine, cysteine,methionine, asparagine or glutamine) can be substituted for each other.Similarly, one or more amino acids comprising aromatic side chains (forexample, phenylalanine, tyrosine or tryptophan) can be substituted foreach other. Similarly, one or more amino acids comprising positivelycharged side chains (for example, lysine, arginine or histidine) can besubstituted for each other. Similarly, one or more amino acidscomprising negatively charged side chains (for example, aspartic acid orglutamic acid) can be substituted for each other. In some embodiments,the modified polymerase is a variant that comprises one or more of theseconservative amino acid substitutions, or any combination thereof. Insome embodiments, conservative substitutions for leucine include:alanine, isoleucine, valine, phenylalanine, tryptophan, methionine, andcysteine. In other embodiments, conservative substitutions forasparagine include: arginine, lysine, aspartate, glutamate, andglutamine.

The term “primer extension activity” and its variants, as used herein,when used in reference to a given polymerase, comprises any in vivo orin vitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing nucleotide incorporation onto the terminal 3′OHend of an extending nucleic acid molecule. Typically but not necessarilysuch nucleotide incorporation occurs in a template-dependent fashion.The primer extension activity is typically quantified as the totalnumber of nucleotides incorporated (as measured by, e.g., radiometric orother suitable assay) by a unit amount of polymerase (in moles) per unittime (seconds) under a particular set of reaction conditions.

The terms “His tag” or “His-tag” and their variants as used hereinrefers to a stretch of amino acids comprising multiple histidineresidues. Typically, the His tag can bind to metal ions, for example,Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺ ions. Optionally, the His tag comprises 2, 3,4, 5, 6, 7, 8 or more histidine residues. In some embodiments, the Histag is fused to the N- or C-terminus of a protein; alternatively, it canbe fused at any suitable location within the protein.

As used herein, the term “biotin moiety” and its variants comprisesbiotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid)and any derivatives and analogs thereof, including biotin-likecompounds. Such compounds include, for example, biotin-e-N-lysine,biocytin hydrazide, amino or sulfhydryl derivatives of 2-iminobiotin andbiotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester,sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide, p-diazobenzoylbiocytin, 3-(N-maleimidopropionyl)biocytin, and the like. “Biotinmoiety” also comprises biotin variants that can specifically bind to anavidin moiety.

The term “biotinylated” and its variants, as used herein, refer to anycovalent or non-covalent adduct of biotin with other moieties such asbiomolecules, e.g., proteins, nucleic acids (including DNA, RNA, DNA/RNAchimeric molecules, nucleic acid analogs and peptide nucleic acids),proteins (including enzymes, peptides and antibodies), carbohydrates,lipids, etc.

The terms “avidin” and “avidin moiety” and their variants, as usedherein, comprises the native egg-white glycoprotein avidin, as well asany derivatives, analogs and other non-native forms of avidin, that canspecifically bind to biotin moieties. In some embodiments, the avidinmoiety can comprise deglycosylated forms of avidin, bacterialstreptavidins produced by selected strains of Streptomyces, e.g.,Streptomyces avidinii, to truncated streptavidins, and to recombinantavidin and streptavidin as well as to derivatives of native,deglycosylated and recombinant avidin and of native, recombinant andtruncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl,N-phthalyl and N-succinyl avidin, and the commercial productsExtrAvidin®, Captavidin®, Neutravidin® and Neutralite Avidin®. All formsof avidin-type molecules, including both native and recombinant avidinand streptavidin as well as derivatized molecules, e.g. nonglycosylatedavidins, N-acyl avidins and truncated streptavidins, are encompassedwithin the terms “avidin” and “avidin moiety”. Typically, but notnecessarily, avidin exists as a tetrameric protein, wherein each of thefour tetramers is capable of binding at least one biotin moiety.

As used herein, the term “biotin-avidin bond” and its variants refers toa specific linkage formed between a biotin moiety and an avidin moiety.Typically, a biotin moiety can bind with high affinity to an avidinmoiety, with a dissociation constant K_(d) typically in the order of10⁻¹⁴ to 10⁻¹⁵ mol/L. Typically, such binding occurs via non-covalentinteractions.

The term “label” and its variants, as used herein, comprises anyoptically detectable moiety and includes any moiety that can be detectedusing, for example, fluorescence, luminescence and/or phosphorescencespectroscopy, Raman scattering, or diffraction. Exemplary labelsaccording to the present disclosure include fluorescent and luminescentmoieties as well as quenchers thereof. Some typical labels includewithout limitation nanoparticles and organic dyes.

The present disclosure relates to compositions and methods comprisingmodified polymerases derived from Phi-29 and/or Phi-29-like phages,wherein the modified polymerases exhibit altered polymerase activity,photostability, label tolerance, kinetics for nucleotide binding and/ornucleotide incorporation kinetics. For example, in some embodiments thedisclosure relates to polymerase compositions, methods of making suchcompositions and methods of using such compositions in variousbiological applications. The compositions and related methods describedherein represent significant advances over the art. For example, in someembodiments, the disclosed compositions and methods can permit nucleicacid synthesis with improved kinetics for real-time visualization ofpolymerase activity. In some embodiments, the disclosed compositions andmethods can permit polymerization of nucleotides (including labelednucleotide analogs) with increased tolerance for the presence of labeledmoieties, for example nanoparticles. In some embodiments, the disclosedcompositions and methods can permit increased primer extension activityin conjunction with use of higher intensities or duration of exposure toexcitation radiation. Such compositions and methods can facilitate, forexample, applications involving nucleic acid synthesis, includingapplications requiring direct monitoring of polymerase activity in vitroor in vivo, such as real-time single molecule sequencing.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a photostability that is at least about 80% understandard photostability assay conditions. In some embodiments, themodified DNA polymerase comprises an amino acid sequence that is atleast about 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 7.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard photostability assay conditions.

In some embodiments, the disclosure relates to a modified DNA polymerasecomprising an amino acid sequence that is at least about 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and further including one or more amino acid mutations that increasethe photostability of the enzyme. In some embodiments, the one or moreamino acid mutations increase the photostability of the modified DNApolymerase by at least about 5%, 10%, 25%, 50%, 75%, 100%, 125%, 250%,500%, 750% or 1000%.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a nanoparticle tolerance that is at least about 80%under standard nanoparticle tolerance assay conditions. In someembodiments, the modified DNA polymerase comprises an amino acidsequence that is at least about 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments, the disclosure relates to a modified DNA polymerasecomprising an amino acid sequence that is at least about 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and further including one or more amino acid mutations that increasethe nanoparticle tolerance of the enzyme. In some embodiments, the oneor more amino acid mutations increase the nanoparticle tolerance of themodified DNA polymerase by at least about 5%, 10%, 25%, 50%, 75%, 100%,125%, 250%, 500%, 750% or 1000%.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard nanoparticle tolerance assay conditions.

In some embodiments, the disclosure relates to a modified DNA polymerasecomprising an amino acid sequence that is at least about 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and further including one or more amino acid mutations that increasethe primer extension activity of the enzyme. In some embodiments, theone or more amino acid mutations increase the primer extension activityof the modified DNA polymerase by at least about 5%, 10%, 25%, 50%, 75%,100%, 125%, 250%, 500%, 750% or 1000%.

In some embodiments, the present disclosure relates to a modified DNApolymerase comprising an amino acid sequence that is at least about 85%,90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified DNA polymerase hasa nanoparticle tolerance that is at least about 80% under standardnanoparticle tolerance assay conditions. In some embodiments, themodified DNA polymerase has a photostability that is at least about 80%under standard photostability assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase having a primer extension activity that is at least about105%, 110%, 125%, 150%, 175%, 200%, 250%, 375%, 500%, 750% or 1000%relative to the primer extension activity of a wild type Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 understandard nanoparticle tolerance assay conditions.

In some embodiments, the present disclosure relates to a modified DNApolymerase comprising an amino acid sequence that is at least about 85%,90%, 95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. The modified DNA polymerases of the presentdisclosure can further comprise one or more amino acid substitutionsselected from the group consisting of: D9A, E11A, E11I, T12I, H58R,N59D, D63A, Y162F, Y162C, D166A, Q377A, S385G, H370G, H370T, H370S,H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T,E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E,K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T,K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H, D507G, D507E,D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H,K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W,K509Y and K509F, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7.

Optionally, the modified DNA polymerases of the present disclosure canfurther comprise a mutation reducing 3′ to 5′ exonuclease activity. Themutation reducing 3′ to 5′ exonuclease activity of the modified DNApolymerase can optionally include one or more amino acid substitutionsat positions selected from the group consisting of: 2, 9, 11, 12, 58,59, 63, 162, 166, 377 and 385. In some embodiments, the one or moreamino acid substitutions can be selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7.

In some embodiments, the modified DNA polymerases of the presentdisclosure can further comprise a mutation increasing the branchingratio of the polymerase in the presence of a labeled nucleotide.Optionally, the modified DNA polymerase comprises an amino acid sequencethat is at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 7, and further comprises one ormore mutations increasing the branching ratio of the modified DNApolymerase. In some embodiments, the one or more mutations comprise oneor more amino acid substitutions at positions selected from the groupconsisting of: 370, 371, 372, 373, 374, 375, 376 and 377. In someembodiments, the one or more mutations can be selected from the groupconsisting of: H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W,H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q,E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q,K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W,K380Y, K380F, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R,D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T,K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modified DNA polymerase comprises an amino acidsequence that is at least about 85%, 90%, 95%, 97% or 99% identical tothe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 and furtherincludes the amino acid substitution H370R. In some embodiments, thepresence of the amino acid substitution H370R increases the branchingratio of the modified polymerase by at least about 5%, 10%, 25%, 50%,75%, 100%, 150%, 200%, 250%, 500%, 750%, 1000% or greater.

In some embodiments, the modified DNA polymerases of the presentdisclosure can further comprise a mutation increasing the primerextension activity. Optionally, the mutation increasing the primerextension activity of the modified DNA polymerase comprises one or moreamino acid substitutions at positions selected from the group consistingof: 129 and 339, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the modified DNA polymerases of the presentdisclosure can further comprise a mutation that increases the nucleotidebinding affinity of the polymerase for a particular labeled nucleotide.Optionally, the mutation increasing the nucleotide binding affinity fora particular labeled nucleotide comprises one or more amino acidsubstitutions at positions selected from the group consisting of: 370,371, 372, 373, 374, 375, 376, 507 and 509. In some embodiments, the oneor more amino acid substitutions can be selected from the groupconsisting of: H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W,H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q,E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q,K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W,K380Y, K380F, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R,D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T,K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the disclosure relates to a nucleic acid moleculeencoding any one, some or all of the modified DNA polymerases of thepresent disclosure. The nucleic acid molecule can optionally be DNA orRNA.

In some embodiments, the disclosure relates to a vector comprising a DNAencoding any one, some or all of the modified DNA polymerases of thepresent disclosure.

In some embodiments, the disclosure relates to an isolated host cellcomprising a vector including a DNA encoding any one, some or all of themodified DNA polymerases of the present disclosure.

In one embodiment, the disclosure relates to compositions and methodsrelating to modified polymerases comprising one or more modificationsrelative to a reference polymerase. In some embodiments, the referencepolymerase is a Phi-29 polymerase comprising the amino acid sequence ofSEQ ID NO: 1 or SEQ ID NO: 20. In some embodiments, the referencepolymerase is a B103 polymerase comprising the amino acid sequence ofSEQ ID NO: 6.

In some embodiments, the modified polymerase is an isolated variant of aB103 polymerase comprising the amino acid sequence of SEQ ID NO: 6,wherein the variant comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and further comprises anyone, two, three or more modifications at amino acid positions 2, 9, 12,14, 15, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374,375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, or any combinationsthereof, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the modifications can includedeletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 7 and comprises any one, two, three or moremutations selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A, S385G, H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H,E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H, D507G,D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F,K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q,K509W, K509Y and K509F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the modifiedpolymerase comprises any one, two, three or more of these amino acidsubstitutions.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, andfurther comprises one or more amino acid substitutions selected from thegroup consisting of: H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, D507H, D507G, D507E, D507T, D507S, D507R, D507A,D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E,K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein thenumbering is relative to the sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises one or moremodifications resulting in an alteration (for example, an increase ordecrease) in polymerase activity relative to the polymerase activity ofa reference polymerase.

In some embodiments, the modified polymerase comprises one or moremodifications resulting in a change in the kinetic behavior of thepolymerase in vitro or in vivo. For example, the modification(s) mayresult in a change (for example, an increase or decrease) in one or moreof the following activities or properties of the polymerase, relative tothe corresponding activity or property of a reference polymerase:specific activity as measured in a primer extension assay; specificactivity as measured in a nucleotide incorporation assay (includingassays for incorporation of naturally occurring nucleotides andnucleotide analogs); exonuclease activity (including, for example, 3′ to5′ exonuclease activity); ability to bind one or more substrates(including naturally occurring nucleotides and nucleotide analogs);yield of synthesized nucleic acid product; processivity; fidelity ofnucleic acid synthesis; rate of nucleic acid synthesis; binding affinityfor one or more particular nucleotides (including naturally occurringnucleotides and nucleotide analogs); K_(m) for one or more substrates(including naturally occurring nucleotides, nucleotide analogs and/ortemplate strand); k_(cat) or V_(max), t_(po1), t⁻¹, k_(po1), or k⁻¹ forone or more nucleotides (including naturally occurring nucleotides andnucleotide analogs); binding affinity for one or more nucleotides(including naturally occurring nucleotides and nucleotide analogs);residence time of one or more nucleotides (including naturally occurringnucleotides and nucleotide analogs) within one or more polymerase activesites; rate of binding for one or more nucleotides (including naturallyoccurring nucleotides and nucleotide analogs); rate of nucleotiderelease (in either altered or unaltered state) from the polymeraseactive site, including, for example, rate of product release; averagetemplate read length in presence of nucleotides (including naturallyoccurring nucleotides and nucleotide analogs); stability of thepolymerase under a given set of conditions, including photostability andchemical stability; tolerance for the presence of labels (including bothorganic labels, e.g., dyes, and inorganic labels, e.g., nanoparticles);and photostability.

In some embodiments, the modification(s) result in an alteration (e.g.,increase or decrease) of any one, some or all of the above activities orproperties or properties relative to the corresponding activity orproperty of a reference polymerase.

The reference polymerase can be any suitable polymerase whose polymeraseactivity can be measured and compared to the activity of a modifiedpolymerase of the present disclosure. In some embodiments, the referencepolymerase can be the unmodified counterpart of the modified polymerase.In some embodiments, the reference polymerase is a naturally occurringpolymerase. The naturally occurring polymerase can in some embodimentsbe derived from Phi-29 or the Phi-29-like family of bacteriophages,including Phi-29, B103 and RB69; alternatively, it can be anon-Phi-29-like polymerase, including, for example, T7 DNA polymerase,Taq DNA polymerase or E. coli DNA polymerase. In some embodiments, thereference polymerase can be a Phi-29 polymerase. (See, for example, U.S.Pat. Nos. 5,001,050; 5,198,543 and 5,576,204 to Blanco et al.). In someembodiments, the reference polymerase is wild type Phi-29 polymerasehaving the amino acid sequence of SEQ ID NO: 1, or a His-tagged versionof Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 20. Insome embodiments, the reference polymerase can be a B103 polymerasecomprising the amino acid sequence of SEQ ID NO: 6. In some embodiments,the reference polymerase can be a variant of a B103 polymerasecomprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the reference polymerase is an RB69 polymerase havingthe amino acid sequence of SEQ ID NO: 15. In some embodiments, thereference polymerase can comprise the amino acid sequence of SEQ ID NO:13. In some embodiments, the reference polymerase can comprise the aminoacid sequence of SEQ ID NO: 14. (See, for example, Meijer et al.,“Phi-29 family of phages” Microbiol. & Mol. Biol. Revs. 65(2):261-287(2001)).

In some embodiments, the reference polymerase is the same polymerase inunmodified form. In some embodiments, the reference polymerase is apolymerase having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:20. In some embodiments, the reference polymerase comprises the aminoacid sequence of SEQ ID NO: 6. In some embodiments, the referencepolymerase is RB69 polymerase comprising the amino acid sequence of SEQID NO: 15.

In some embodiments, the modified polymerase exhibits an increase ordecrease in the branching ratio in the presence of a particularnucleotide, for example, a labeled nucleotide analog, relative to theunmodified polymerase, or relative to any other reference polymerasesuch as, for example, a Phi-29 polymerase having the amino acid sequenceof SEQ ID NO: 1.

In some embodiments, the modified polymerase exhibits an increase in thet⁻¹ value for a particular nucleotide, for example, a labeled nucleotideanalog, relative to an unmodified counterpart.

In some embodiments, the modified polymerase exhibits a t⁻¹ value for alabeled nucleotide that is equal to or greater than the t⁻¹ value forthe same nucleotide of a reference Phi-29 polymerase comprising theamino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase exhibits an increase in thet_(po1) value for a particular nucleotide, for example, a labelednucleotide analog, relative to an unmodified counterpart.

In some embodiments, the modified polymerase exhibits a t_(po1) valuefor a labeled nucleotide that is equal to or lesser than the t_(po1)value for the same nucleotide of a reference Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase exhibits an increase inresidence time for a particular nucleotide, for example, a labelednucleotide analog, relative to an unmodified counterpart.

In some embodiments, the modified polymerase exhibits a residence timefor a labeled nucleotide that is equal to or greater than the residencetime for the same nucleotide of a reference Phi-29 polymerase comprisingthe amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase exhibits an increase inphotostability relative to an unmodified counterpart.

In some embodiments, the modified polymerase exhibits a photostabilitythat is greater than the photostability of a reference Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase exhibits an increase innanoparticle tolerance relative to an unmodified counterpart.

In some embodiments, the modified polymerase exhibits a nanoparticletolerance that is greater than the nanoparticle tolerance of a referencePhi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase is operably linked to adetectable label. In some embodiments, the detectable label is ananoparticle.

Also provided herein are polynucleotides encoding the modifiedpolymerases of the present disclosure, and isolated host cellscomprising one or more of these polynucleotides.

Also provided herein are vectors comprising one or more polynucleotidesencoding the modified polymerases of the present disclosure, andisolated host cells comprising these vectors. In some embodiments, thevectors further comprise a promoter operably linked to thepolynucleotide encoding the modified polymerase. The promoter can beconstitutive or inducible. The host cell comprising the polynucleotideencoding the modified polymerase can be eukaryotic or prokaryotic.

Also provided are methods for producing one or more modified polymerasesof the present disclosure, comprising: culturing a host cell comprisinga polynucleotide that encodes a modified polymerase under conditionsthat are suitable for the host cell to produce the active modifiedpolymerase. Optionally, the method can include the step of purifying themodified polymerase from the isolated host cells. Also disclosed hereinis an active modified polymerase produced by this method.

Also provided herein are methods for nucleotide incorporation,comprising: contacting a modified polymerase of the present disclosurewith a target nucleic acid molecule and with at least one nucleotideunder conditions where the at least one labeled nucleotide isincorporated by the modified polymerase into an extending nucleic acidmolecule. In some embodiments, the modified polymerase can comprise anamino acid sequence at least 90%, 95%, 97,%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In someembodiments, the at least one nucleotide can be a labeled nucleotide. Insome embodiments, the labeled nucleotide comprises a nucleotide operablylinked to a detectable label, referred to herein as a nucleotide label.In some embodiments, the nucleotide label can emits one or more signalsindicative of incorporation of the labeled nucleotide. In someembodiments, the method can further comprise the step of detecting theincorporation of the labeled nucleotide by detecting the one or moresignals emitted by the nucleotide label. In some embodiments, theincorporation is a productive or non-productive incorporation.

Also provided herein is a method for determining a nucleotide sequenceof a single nucleic acid molecule, comprising: (a) conducting anucleotide polymerization reaction comprising a target nucleic acidmolecule, a modified polymerase and at least one labeled nucleotide,which reaction results in the incorporation of one or more labelednucleotides by the modified polymerase and the generation of one or moredetectable signals indicative of one or more nucleotide incorporations;(b) detecting a time sequence of nucleotide incorporations; and (c)determining the identity of one or more incorporated nucleotides,thereby determining some or all of the nucleotide sequence of the targetnucleic acid molecule. In some embodiments, the determination ofnucleotide sequence can occur in real or near real time. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 8, and can optionally further includeone or more amino acid mutations selected from the group consisting of:T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y,T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y,H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W,E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W,K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y,K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q,A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R,D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T,K509S, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit increase branching ratioand/or increased photostability and/or increased nanoparticle tolerancerelative to a reference polymerase having the amino acid sequence of SEQID NO: 7. Optionally, the modified polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 1, and can optionally further include an amino acid substitution atposition 383, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 1.

Also disclosed herein is a method for determining a nucleotide sequenceof a single nucleic acid molecule, comprising the steps of: (a)conducting a polymerase reaction comprising a polymerase and at leastone labeled nucleotide, such that one or more labeled nucleotidesinteract successively with the polymerase, thereby generating or causingto be generated one or more detectable signals indicative of one or morepolymerase-nucleotide interactions, and where the polymerase comprisesthe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8; (b) detecting atime sequence of polymerase-nucleotide interactions; and (c) determiningthe identity of the one or more labeled nucleotides that interact withthe polymerase during the one or more polymerase-nucleotideinteractions, thereby determining some or all of the nucleotide sequenceof a single nucleic acid molecule. In some embodiments, thedetermination of nucleotide sequence can occur in real or near realtime. In some embodiments, the modified polymerase can comprise an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 8. In someembodiments, the modified polymerase can further comprise one or moreamino acid substitutions selected from the group consisting of: T365G,T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H,H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F,E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y,E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W,A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q,D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S,K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. Typically, thismodified polymerase can exhibit increase branching ratio and/orincreased photostability and/or increased nanoparticle tolerancerelative to a reference polymerase having the amino acid sequence of SEQID NO: 7. Optionally, the modified polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 1, and further comprise one ormore amino acid substitutions increasing the branching ratio of theenzyme. Optionally, the one or more amino acid substitutions can includereplacement of the lysine residue at position 373 of SEQ ID NO: 1 withany amino acid other than lysine.

In some embodiments of the methods of the present disclosure, themodified polymerase can exhibit a t⁻¹ value for a labeled nucleotidethat is equal to or greater than the t⁻¹ value for the same nucleotideof a reference Phi-29 polymerase comprising the amino acid sequence ofSEQ ID NO: 1.

In some embodiments of the methods of the present disclosure, themodified polymerase can exhibit a t_(po1) value for a labeled nucleotidethat is equal to or lesser than the t_(po1) value for the samenucleotide of a reference Phi-29 polymerase comprising the amino acidsequence of SEQ ID NO: 1.

In some embodiments of the methods of the present disclosure, themodified polymerase can exhibit a residence time for a labelednucleotide that is equal to or greater than the residence time for thesame nucleotide of a reference Phi-29 polymerase comprising the aminoacid sequence of SEQ ID NO: 1.

In some embodiments of the methods of the present disclosure, themodified polymerase can exhibit a photostability that is equal to orgreater than the photostability of a reference Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments of the methods of the present disclosure, themodified polymerase can exhibit a nanoparticle tolerance that is equalto or greater than the nanoparticle tolerance of a reference Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments of the methods of the present disclosure, thelabeled nucleotide can comprise a nucleotide label operably linked tothe nucleotide. In some embodiments, the nucleotide label can beoperably linked to the base, sugar or phosphate group of the nucleotide.

In some embodiments of the methods of the present disclosure, thelabeled nucleotide can be a reversible terminator. In some embodiments,the modified polymerase can be operably linked to a detectable label. Insome embodiments, the label of the modified polymerase and thenucleotide label can be capable of undergoing FRET with each other.Typically, FRET between the polymerase label and the nucleotide labelresults in the emission of one or more FRET signals. Optionally, the oneor more FRET signals can be detected and analyzed to determine theoccurrence of a polymerase-nucleotide interaction, or the base identityof the underlying nucleotide.

Although the polymerases differ from organism to organism, suchpolymerases are typically capable of template-dependent nucleic acidsynthesis and share several highly conserved domains. Various studieshave examined the phylogenetic relationships among various polymerases.See, e.g., Bernad, A., et al., “Structural and functional relationshipsbetween prokaryotic and eukaryotic DNA polymerases” EMBO J.6(13):4219-4225 (1987); Braithwaite, D. K. & Ito, J., “Compilation,alignment, and phylogenetic relationships of DNA polymerases” Nucl.Acids Res. 21:787-802 (1993); Steitz et al., “DNA polymerases:structural diversity and common mechanisms” J. Biol. Chem.274:17395-17398 (1999). Based on such studies, polymerases have beenclassified into the Family A DNA polymerases (based on homology to theproduct of the polA gene encoding E. coli DNA polymerase I); the FamilyB DNA polymerases (based on homology to the product of the polB geneencoding E. coli DNA polymerase II); and the Family C DNA polymerases(based on homology to the product of the polC gene encoding E. coli DNApolymerase III alpha subunit). The Family B DNA polymerases can includethe DNA polymerase of the bacteriophage Phi-29, (also known as D29 orphi29) of the Podoviridae family of phages.

In some embodiments, the modified polymerase can be a modifiedPhi-29-like polymerase comprising an amino acid sequence that is atleast 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence ofSEQ ID NO: 1:

(SEQ ID NO: 1) MKHMPRKMYS CDFETTTKVE DCRVWAYGYM NIEDHSEYKI        70         80         90        100GNSLDEFMAW VLKVQADLYF HNLKFDGAFI INWLERNGFK       110        120        130        140WSADGLPNTY NTIISRMGQW YMIDICLGYK GKRKIHTVIY       150        160        170        180DSLKKLPFPV KKIAKDFKLT VLKGDIDYHK ERPVGYKITP       190        200        210        220EEYAYIKNDI QIIAEALLIQ FKQGLDRMTA GSDSLKGFKD       230        240        250        260IITTKKFKKV FPTLSLGLDK EVRYAYRGGF TWLNDRFKEK       270        280        290        300EIGEGMVFDV NSLYPAQMYS RLLPYGEPIV FEGKYVWDED       310        320        330        340YPLHIQHIRC EFELKEGYIP TIQIKRSRFY KGNEYLKSSG       350        360        370        380GEIADLWLSN VDLELMKEHY DLYNVEYISG LKFKATTGLF       390        400        410        420KDFIDKWTYI KTTSEGAIKQ LAKLMLNSLY GKFASNPDVT       430        440        450        460GKVPYLKENG ALGFRLGEEE TKDPVYTPMG VFITAWARYT       470        480        490        500TITAAQACYD RIIYCDTDSI HLTGTEIPDV IKDIVDPKKL       510        520        530        540GYWAHESTFK RAKYLRQKTY IQDIYMKEVD GKLVEGSPDD       550        560        570YTDIKFSVKC AGMTDKIKKE VTFENFKVGF SRKMKPKPVQ         VPGGVVLVDD TFTIK

In some embodiments, the reference polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the polymerase can be a mutant Phi-29 polymerasethat retains nucleotide polymerization activity but lacks the 3′→5′ or5′→3′ exonuclease activity. For example, mutant Phi-29 polymeraseshaving exonuclease-minus activity, or reduced exonuclease activity, caninclude the amino acid sequence of SEQ ID NO: 1 and further comprise oneor more amino acid substitutions at positions selected from the groupconsisting of: 12, 14, 15, 62, 66, 165 and 169 (wherein the numbering isrelative to the amino acid sequence of wild type Phi-29). In someembodiments, the polymerase is a phi29 polymerase comprising the aminoacid sequence of SEQ ID NO: 1 and one or more of the following aminoacid substitutions: D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C,and D169A, wherein the numbering is relative to SEQ ID NO: 1. In someembodiments, the reference polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further comprise one ormore of the following amino acid substitutions: D12A, E14I, E14A, T15I,N62D, D66A, Y165F, Y165C, and D169A, wherein the numbering is relativeto SEQ ID NO: 1. In some embodiments, the polymerase is a phi29polymerase comprising the amino acid sequence of SEQ ID NO: 1 and one orboth of the following amino acid substitutions: D12A and D66A. In someembodiments, the reference polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further comprise one orboth of the following amino acid substitutions: D12A and D66A, whereinthe numbering is relative to SEQ ID NO: 1. See, e.g., Blanco, U.S. Pat.Nos. 5,001,050, 5,198,543, and 5,576,204; and Hardin PCT/US2009/31027with an International filing date of Jan. 14, 2009. Optionally, thepolymerase can further include a biotinylation site or His tag asdescribed herein.

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further include one ormore amino acid mutations at positions selected from the groupconsisting of: 132, 135, 250, 266, 332, 342, 368, 370, 371, 372, 373,375, 379, 380, 383, 387, 390, 458, 478, 480, 484, 486, and 512, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 1. Insome embodiments, the modified polymerase can comprise an amino aciddeletion, wherein the deletion includes some of all of the amino acidsspanning positions 306 to 311.

In some embodiments, the reference polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further include one ormore amino acid mutations at positions selected from the groupconsisting of: 132, 135, 250, 266, 332, 342, 368, 371, 375, 379, 380,383, 387, 390, 458, 478, 480, 484, 486, 510 and 512, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further includes one ormore amino acid mutations selected from the group consisting of: K132A,K135A, K135D, K135E, V250A, V250C, Y266F, D332Y, L342G, T368D, T368E,T368F, K370A, K371E, T372D, T372E, T372R, T372K, E375A, E375F, E375H,E375K, E375Q, E375R, E375S, E375W, E375Y, K379A, Q380A, K383E, K383H,K383L, K383R, N387Y, Y390F, D458N, K478D, K478E, K478R, L480K, L480R,A484E, E486A, E486D, K512A K512D, K512E, K512R, K512Y,K371E/K383E/N387Y/D458N, Y266F/Y390F, Y266F/Y390F/K379A/Q380A,K379A/Q380A, E375Y/Q380A/K383R, E375Y/Q380A/K383H, E375Y/Q380A/K383L,E375Y/Q380A/V250A, E375Y/Q380A/V250C, E375Y/K512Y/T368F,E375Y/K512Y/T368F/A484E, K379A/E375Y, K379A/K383R, K379A/K383H,K379A/K383L, K379A/Q380A, V250A/K379A, V250A/K379A/Q380A,V250C/K379A/Q380A, K132A/K379A and deletion of some or all of the aminoacid residues spanning R306 to K311, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 1.

Also disclosed herein is a method for single molecule sequencing in realtime, comprising: contacting a modified Phi-29 polymerase according tothe present disclosure with labeled nucleotide under conditions wherethe labeled nucleotides are incorporated by the polymerase onto the endof an extending nucleic acid molecule, accompanied by the emission of asignal indicative of a nucleotide incorporation; detecting a timesequence of nucleotide incorporations; and analyzing the time sequenceof nucleotide incorporations to determine a polynucleotide sequence.

In some embodiments, the reference polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 1 and further includes one ormore amino acid mutations selected from the group consisting of: K132A,K135A, K135D, K135E, V250A, V250C, Y266F, D332Y, L342G, T368D, T368E,T368F, K370A, K371E, T372D, T372E, T372R, T372K, T373A, T373F, T373H,T373K, T373Q, T373R, T373S, T373W, T373Y, T373E, E375A, E375F, E375H,E375K, E375Q, E375R, E375S, E375W, E375Y, K379A, Q380A, K383E, K383H,K383L, K383R, N387Y, Y390F, D458N, K478D, K478E, K478R, L480K, L480R,A484E, E486A, E486D, K510A, K510F, K510H, K510K, K510Q, K510R, K510S,K510W, K510Y, K510E, K512A K512D, K512E, K512R, K512Y,K371E/K383E/N387Y/D458N, Y266F/Y390F, Y266F/Y390F/K379A/Q380A,K379A/Q380A, E375Y/Q380A/K383R, E375Y/Q380A/K383H, E375Y/Q380A/K383L,E375Y/Q380A/V250A, E375Y/Q380A/V250C, E375Y/K512Y/T368F,E375Y/K512Y/T368F/A484E, K379A/E375Y, K379A/K383R, K379A/K383H,K379A/K383L, K379A/Q380A, V250A/K379A, V250A/K379A/Q380A,V250C/K379A/Q380A, K132A/K379A and deletion of some or all of the aminoacid residues spanning R306 to K311, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 1.

Without being bound to any particular theory, it is thought that thedomain comprising amino acid residues 304-314 of the amino acid sequenceof SEQ ID NO: 3 (Phi-29 polymerase), or homologs thereof, can reduce orotherwise interfere with DNA initiation and/or elongation by inhibitingaccess to the Phi-29 polymerase active site, and that this region mustbe displaced in order to allow access to the active site. See, e.g.,Kamtekar et al., “The Φ29 DNA polymerase:protein primer structuresuggests a model for the initiation to elongation transition”, EMBO J.,25:1335-1343 (2005).

In some embodiments, the modified polymerase is derived from apolymerase of any member of the Phi-29-like family of phages. ThePhi-29-like phages are a genus of phages that are related to Phi-29 thatincludes the phages PZA, Φ15, BS32, B103, M2Y (M2), Nf1 and GA-1. Phagesof this group have been sub-classified into three groups based onserological properties, DNA and/or polymerase maps and partial orcomplete DNA sequences, and share several characteristics in common. Forexample, such phages can typically undergo protein-primed DNAreplication. See, for example, Meijer et al., “Phi-29 family of phages”Microbiol. & Mol. Biol. Revs. 65(2):261-287 (2001).

The genome of the Phi-29-like phage B103, including a gene encoding aB103 DNA polymerase, has been sequenced. See, e.g., Pecenkova et al.,“Bacteriophage B103: complete DNA sequence of its genome andrelationship to other Bacillus phages” Gene 199:157-163 (1999). The DNApolymerase of B103 is homologous to the DNA polymerase of Phi-29 and ofother Phi-29-like phages. Collectively, these polymerases share severalhighly conserved regions. See, e.g., Meijer et al., “Phi-29 family ofphages” Microbiol. & Mol. Biol. Revs. 65(2):261-287 (2001). Theseconserved regions are typically characterized by several conserved aminoacid motifs. See, e.g., Blanco et al., Gene 100:27-38 (1991); Blasco etal., “Φ29 DNA polymerase Active Site” J. Biol. Chem. 268:16763-16770(1993) (describing regions of sequence homology and mutational analysisof consensus regions of Phi-29 and Phi-29-like DNA polymerases); Bermanet al., “Structures of phi29 DNA polymerase complexed with substrate:the mechanism of translocation in B-family polymerases”, EMBO J.,26:3494-3505 (2007). Site-directed mutagenesis indicates that thesethree regions can include an evolutionarily conserved polymerase activesite.

Collectively, the polymerases of these phages share several highlyconserved regions. See, e.g., Meijer et al., “Phi-29 family of phages”Microbiol. & Mol. Biol. Revs. 65(2):261-287 (2001). The conservedregions are typically characterized by the following amino acid motifs:

(SEQ ID NO: 2) Dx₂SLYP 

The consensus sequence of SEQ ID NO: 2 represents the consensus aminoacid sequence for a motif known as region 1, also named motif A.

(SEQ ID NO: 3) Kx₃NSxYG

The consensus sequence of SEQ ID NO: 3 represents the consensus aminoacid sequence for the motif known as region 2a, also named motif B.

(SEQ ID NO: 4) YGDTDS

The consensus sequence of SEQ ID NO: 4 represents the consensus aminoacid sequence for the motif known as region 3, also named motif C.

(SEQ ID NO: 5) KxY 

The consensus sequence of SEQ ID NO: 5 represents the consensus aminoacid sequence for the motif known as region 4. See, e.g., Blanco et al.,Gene 100:27-38 (1991); Blasco et al., “Φ29 DNA polymerase Active Site”J. Biol. Chem. 268:16763-16770 (1993) (describing regions of sequencehomology and mutational analysis of consensus regions of Phi-29 andPhi-29-like DNA polymerases).

Site-directed mutagenesis indicates that these motifs can include one ormore evolutionarily conserved polymerase active sites.

In some embodiments, the modified polymerase is

In some embodiments, the modified polymerase is derived from aPhi-29-like polymerase and comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 6 as follows:

(SEQ ID NO: 6) 1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl 61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmfdslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified polymerase is derived from apolymerase of the Phi-29-like phages and comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence, wherein the modified polymerasecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, orany biologically active fragment thereof.

In some embodiments, the modified polymerase is homologous to apolymerase of one or more of the following organisms: B103, Phi-29,GA-1, PZA, Phi-15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7,PR4, PR5, PR722, or L17. See, e.g., Meijer et al., “Phi-29 family ofphages,” Microbiol. & Mol. Biol. Revs. 65(2):261-287 (2001).

In some embodiments, the modified polymerase is derived from a B103polymerase having the amino acid sequence of SEQ ID NO: 6 and furthercomprises one or more mutations in the amino acid sequence of SEQ ID NO:6. In some embodiments, the one or more mutations can include, forexample, substitution, chemical modification, addition, deletion and/orinversion of one or more amino acid residues, or any combination of theforegoing.

For example, in some embodiments, the modified polymerase comprises theamino acid sequence of SEQ ID NO: 6, and further comprises one or moreamino acid mutations (e.g., amino acid substitutions, additions,deletions) that increases polymerase activity, reduces 3′ to 5′exonuclease activity, increases the branching ratio of the polymerasewith a particular labeled nucleotide, and/or increases the residencetime of a particular labeled nucleotide within the polymerase activesite. The modification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications.

For example, in some embodiments, the modified polymerase includes oneor more amino acid mutations that increase the polymerase activity ofthe modified polymerase relative to a reference polymerase having theamino acid sequence of SEQ ID NO: 6. For example, in some embodiments,the modified polymerase comprises an amino acid mutation at position383, at position 384, or at both positions 383 and 384, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises the amino acidsubstitutions F383L and D384N, and exhibits increased polymeraseactivity relative to the reference polymerase having the amino acidsequence of SEQ ID NO: 6. Typically, such mutants exhibit increasedpolymerase activity relative to the unmodified protein having the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase having increased polymeraseactivity relative to the reference polymerase of SEQ ID NO: 6 comprisesan amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 6, or anybiologically active fragment thereof, wherein the amino acid at position383 is not phenylalanine (F), where the numbering is relative to theamino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 6, and further comprises an amino acidsubstitution at position 383, wherein the numbering is relative to aB103 polymerase having the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6, or any biologically active fragment thereof, wherein the modifiedpolymerase further comprises the amino acid mutation F383L.

In some embodiments, the modified polymerase is a variant of a B103polymerase that comprises an amino acid sequence that is at least 80%,85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 6, or any biologically active fragment thereof, wherein theamino acid at position 384 is not aspartic acid (D), where the numberingis relative to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 6 and further comprises an amino acidsubstitution at position 384, wherein the numbering is relative to aB103 polymerase having the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 80%, 85%, 90%,95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6, or any biologically active fragment thereof, wherein the modifiedpolymerase further comprises the amino acid mutation D384N.

In some embodiments, the modified polymerase is a variant of B103polymerase, or any biologically active fragment thereof, having theamino acid sequence of SEQ ID NO: 6, wherein the modified variantfurther comprises amino acid substitutions at positions 383 and 384,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the modified polymerase comprises the aminoacid substitutions F383L and D384N, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 6. The amino acid sequence of thismodified polymerase can be represented as follows:

(SEQ ID NO: 7) 1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl 61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified B103 polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 7, or anybiologically active fragment thereof. Typically, a modified polymerasehaving the amino acid sequence of SEQ ID NO: 7 will exhibit increasedpolymerase activity (e.g., primer extension activity) relative to theunmodified reference polymerase having the amino acid sequence of SEQ IDNO: 6.

In some embodiments, the modified polymerase comprises one or moremodifications resulting in altered exonuclease activity (for example 3′to 5′ exonuclease activity) as compared to a reference polymerase (forexample, an unmodified counterpart). In some embodiments, themodification comprises an amino acid substitution. In some embodiments,the modified polymerase lacks 3′ to 5′ exonuclease activity, or lacks 5′to 3′ exonuclease activity, or both.

Mutations that reduce or eliminate 3′ to 5′ exonuclease activity havebeen described, for example, in Phi-29 polymerase at various residues.See, e.g., de Vega et al., “Primer-terminus stabilization at the 3′-5′exonuclease active site of Φ29 DNA polymerase. Involvement of two aminoacid residues highly conserved in proofreading DNA polymerases” EMBO J.,15(5):1182-1192 (1996); Soengas et al., “Site-directed mutagenesis atthe Exo III motif of Φ29 DNA polymerase; overlapping structural domainsfor the 3′-5′ exonuclease and strand-displacement activities” EMBO J.,11(11):4227-4237 (1992); Blanco et al., U.S. Pat. Nos. 5,001,050,5,198,543 and 5,576,204.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions, additions or deletionsat one or more positions selected from the group consisting of: 2, 9,11, 12, 58, 59, 63, 162, 166, 377 and 385, wherein the numbering isrelative to SEQ ID NO: 6. In some embodiments, this modified polymerasecan exhibit reduced exonuclease activity relative to an unmodifiedcounterpart.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6 or SEQ ID NO: 7, or toany biologically active fragment thereof, and further comprises theamino acid mutation D166A, wherein the numbering is relative to SEQ IDNO: 6. In some embodiments, this modified polymerase can exhibit reduced3′ to 5′ exonuclease activity relative to a reference polymerase havingthe amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to SEQ ID NO:6. In some embodiments, this modified polymerase can exhibit reducedexonuclease activity relative to an unmodified counterpart.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,Q377A and S385G, wherein the numbering is relative to SEQ ID NO: 6.Typically, this modified polymerase can exhibit reduced 3′ to 5′exonuclease activity relative to an unmodified counterpart.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 6 and further comprises an amino acidsubstitution wherein the amino acid residue at position 9 is replacedwith an alanine (“A”) residue, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 6 and further comprises one or more of theamino acid substitutions D9A, E11A, T12I, H58R, N59D, D63A, D166A,Q377A, S385G, or any combination thereof, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modified polymerase comprises any one, two, three,four, five or all of these mutations. In some embodiments, the modifiedpolymerase comprises the amino acid substitutions D9A and D63A. In someembodiments, the modified polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, this polymerase will exhibitreduced 3′ to 5′ exonuclease activity relative to an unmodifiedcounterpart.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 andcomprises any one, two, three or more of the mutations described herein.

Optionally, the modification(s) reducing 3′ to 5′ exonuclease activitycan be combined with additional modification(s) that increase polymeraseactivity. Modifications increasing polymerase activity include, forexample, the amino acid substitutions F383L and D384N.

In one embodiment, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 6 and further comprises the amino acidsubstitution D166A, which reduces the 3′ to 5′ exonuclease activity, incombination with the amino acid substitutions F383L and D384N, whichincrease the polymerase activity of the modified polymerase, relative tothe unmodified protein having the amino acid sequence of SEQ ID NO: 6.The amino acid sequence of this triple mutant polymerase is the aminoacid sequence of SEQ ID NO: 8, below:

(SEQ ID NO: 8) 1mprkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl 61kfdgafivnw lehhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhaerp vgheitpeey eyiknaieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir rayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgaep velyltnvdl eliqehyemy nveyidgfkf rektglfkef 361idkwtyvkth ekgakkqlak lmlnslygkf asnpdvtgkv pylkedgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyakevdgkl iecspdeatt tkfsvkcagm tdtikkkvtf 541dnfrvgfsst gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 8, or anybiologically active fragment thereof. Typically, the modified polymeraseof SEQ ID NO: 8 will exhibit reduced exonuclease activity relative to areference polymerase comprising the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more modifications resulting in altered (e.g.,increased or decreased) branching ratio and/or nucleotide bindingaffinity (K_(D)) as compared to a reference polymerase. For example,mutations that may affect branching ratio and/or nucleotide bindingaffinity can include mutations (e.g., amino acid substitutions,additions or deletions) at positions selected from the group consistingof: 370, 371, 372, 373, 374, 375, 376 and 377, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the mutation comprises one or more of the amino acidsubstitutions selected from the group consisting of H370G, H370T, H370S,H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T,E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E,K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T,K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H, D507G, D507E,D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H,K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W,K509Y and K509F, wherein the numbering is relative to the sequence ofSEQ ID NO: 6.

In some embodiments, the polymerase further comprises one or moremutations reducing the exonuclease activity as described herein such as,for example, the amino acid substitution D166A.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more modifications resulting in altered (e.g.,increased or decreased) primer extension activity as compared to areference polymerase. For example, mutations that may affect primerextension activity can include mutations (e.g., amino acidsubstitutions, additions or deletions) at positions selected from thegroup consisting of: 129 and 339, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation comprises one or more of the amino acid substitutions selectedfrom the group consisting of: In some embodiments, the polymerasefurther comprises one or more mutations reducing the exonucleaseactivity as described herein such as, for example, the amino acidsubstitution D166A.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 75%, 85%, 90%, 95% or 99% identical toany one of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQID NO: 8, or to any biologically active fragment thereof, and furthercomprises one or more modifications resulting in altered (e.g.,increased or decreased) t_(po1) or k_(po1) values as compared to areference polymerase. For example, mutations that may affect t_(po1) ork_(po1) values can include mutations (e.g., amino acid substitutions,additions or deletions) at position 380, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the mutation comprises one or more of the amino acidsubstitutions selected from the group consisting of: K380E, K380T,K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, and results in anincreased t_(po1) value (or decreased k_(po1) value) relative to theunmodified counterpart. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A.

In some embodiments, the modified polymerase is a variant of a B103polymerase comprising the amino acid sequence of SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO: 8, wherein the variant further comprises any one,two, three or more modifications at amino acid positions 2, 9, 12, 14,15, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375,376, 377, 380, 383, 384, 385, 455, 507, 509, or any combinationsthereof, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. In some embodiments, the modifications can includedeletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions.

In some embodiments, the modified polymerase is a variant of a B103polymerase comprising the amino acid sequence of SEQ ID NO: 6, SEQ IDNO: 7 or SEQ ID NO: 8, wherein the variant further comprises any one,two, three or more modifications at amino acid positions selected fromthe group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 98, 129, 166,176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256, 300, 302,310, 339, 357, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509, 511, 526,528, 529, 531, 535, 552, 555, 567, 569 and 572, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this modified polymerase comprises the aminoacid substitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio, increased nucleotide binding affinity, increasedphotostability and/or increased nanoparticle tolerance relative to areference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions at any one, two, three or more positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises amodification at residue at position 2, wherein the numbering is relativeto a B103 polymerase having the amino acid sequence of SEQ ID NO: 6. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the modified polymerase comprises a proline (P) residue atposition 2.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 370, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the amino acid at position 370 is any amino acid otherthan threonine (T) or histidine (H). In some embodiments, the amino acidat position 370 of the modified polymerase is glutamic acid (E), serine(S), lysine (K), arginine (R), alanine (A), glutamine (Q), tryptophan(W), tyrosine (Y), phenylalanine (F) or any other natural or non-naturalamino acid, other than threonine (T). modified polymerase is a variantof B103 polymerase that comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any oneof the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8,or to any biologically active fragment thereof, and further comprises anamino acid substitution selected from the group consisting of: H370E,H370K, H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y andH370F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 371, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 7. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acidsequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or to anybiologically active fragment thereof, wherein the amino acid at position371 is any amino acid other than serine (S). In some embodiments, themodified polymerase comprises a glutamic acid (E) residue at position371. In some embodiments, the amino acid at position 371 is any aminoacid other than serine (S) or glutamic acid (E). In some embodiments,the amino acid at position 371 of the modified polymerase is glycine(G), histidine (H), lysine (K), arginine (R), alanine (A), glutamine(Q), tryptophan (W), tyrosine (Y), phenylalanine (F) or any othernatural or non-natural amino acid, other than serine (S). In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acidsequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or to anybiologically active fragment thereof, and further comprises one or moreamino acid substitutions selected from the group consisting of E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y and E371F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 372, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 7. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acidsequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or to anybiologically active fragment thereof, wherein the amino acid at position372 is any amino acid other than glutamic acid (E). In some embodiments,the modified polymerase comprises a lysine (K) residue at position 372.In some embodiments, the amino acid at position 372 is any amino acidother than glutamic acid (E) or lysine (K). In some embodiments, theamino acid at position 372 of the modified polymerase is glycine (G),histidine (H), serine (S), arginine (R), alanine (A), glutamine (Q),tryptophan (W), tyrosine (Y), phenylalanine (F) or any other natural ornon-natural amino acid, other than glutamic acid (E). In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acidsequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or to anybiologically active fragment thereof, and further comprises one or moreamino acid substitutions selected from the group consisting of: K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y and K372F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 380, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 7. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the amino acid at position 380 can be any amino acid otherthan lysine (K). In some embodiments, the amino acid at position 380 ofthe modified polymerase is glycine (G), histidine (H), serine (S),arginine (R), alanine (A), glutamine (Q), tryptophan (W), tyrosine (Y),phenylalanine (F) or any other natural or non-natural amino acid, otherthan lysine (K). In some embodiments, the modified polymerase is avariant of B103 polymerase that comprises an amino acid sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to anyone of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ IDNO: 8, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Yand K380F, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 6.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 507, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 7. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the amino acid at position 507 can be any amino acid otherthan aspartic acid (D). In some embodiments, the modified polymerasecomprises a histidine (H) residue at position D. In some embodiments,the amino acid at position 380 of the modified polymerase is glycine(G), histidine (H), serine (S), arginine (R), alanine (A), glutamine(Q), tryptophan (W), tyrosine (Y), phenylalanine (F) or any othernatural or non-natural amino acid, other than aspartic acid (D). In someembodiments, the modified polymerase is a variant of B103 polymerasethat comprises an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% identical to any one of the amino acidsequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or to anybiologically active fragment thereof, and further comprises one or moreamino acid substitutions selected from the group consisting of: D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y andD507F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6.

In some embodiments, the modified polymerase is a variant of B103polymerase that comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of theamino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or toany biologically active fragment thereof, and further comprises an aminoacid modification at position 509, wherein the numbering is relative toa B103 polymerase having the amino acid sequence of SEQ ID NO: 7. Themodification can include, for example, one or more amino acidsubstitutions, additions, deletions or chemical modifications. In someembodiments, the amino acid at position 509 can be any amino acid otherthan lysine (K). In some embodiments, the amino acid at position 509 ofthe modified polymerase is glycine (G), histidine (H), serine (S),arginine (R), alanine (A), glutamine (Q), tryptophan (W), tyrosine (Y),phenylalanine (F) or any other natural or non-natural amino acid, otherthan lysine (K). In some embodiments, the modified polymerase is avariant of B103 polymerase that comprises an amino acid sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to anyone of the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 7 and SEQ IDNO: 8, or to any biologically active fragment thereof, and furthercomprises one or more amino acid substitutions selected from the groupconsisting of: K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R,K509A, K509Q, K509W, K509Y and K509F, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 andcomprises any one, two, three or more of the mutations selected from thegroup consisting of: D9A, E11A, T12I, H58R, N59D, D63A, D166A, Q377A,S385G, T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W,T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W,H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q,E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q,K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W,K380Y, K380F, F383L, D384N, A481E, A481F, A481G, A481S, A481R, A481K,A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S,D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D,K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6, 7 or 8, respectively.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, andfurther includes one or more amino acid substitutions selected from thegroup consisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A,T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A,H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R,E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R,K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A,K380Q, K380W, K380Y, K380F, F383L, D384N, A481E, A481F, A481G, A481S,A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E,D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H,K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W,K509Y and K509F, wherein the numbering is relative to the sequence ofSEQ ID NO: 6.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,T12I, H58R, N59D, D63A, D166A, Q377A and S385G, and further comprisesamino acid substitutions at two or more positions selected from thegroup consisting of: 370, 372 and 507.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,T12I, H58R, N59D, D63A, D166A, Q377A and S385G, and further comprisesthe amino acid substitutions K372Y and D507H.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,T12I, H58R, N59D, D63A, D166A, Q377A and S385G, and further comprisesthe amino acid substitutions H370R and D507H.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,T12I, H58R, N59D, D63A, D166A, Q377A and S385G, and further comprisestwo or more amino acid substitutions selected from the group consistingof: H370R, K372Y and D507H.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 7 and further comprises an aminoacid mutation at one, two, three or more amino acid positions selectedfrom the group consisting of: 9, 11, 12, 58, 59, 63, 162, 162, 166, 377and 385, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. Typically, such a modified polymerase will exhibit reduced3′ to 5′ exonuclease activity relative to reference polymerase havingthe amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 7 and further comprises one, two,three or more amino acid mutations selected from the group consistingof: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377Aand S385G, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 6. In some embodiments, the modified polymerase comprisesany one, two, three, four, five or all of these mutations. In someembodiments, the modified polymerase comprises the amino acidsubstitution D166A. In some embodiments, the modified polymerasecomprises the amino acid substitutions D9A and D63A. In someembodiments, the modified polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, such modified polymerases willexhibit reduced 3′ to 5′ exonuclease activity relative to referencepolymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furthercomprises an amino acid substitution at one or more positions selectedfrom the group consisting of: 2, 73, 107, 147, 221, 318, 339, 359, 372,405, 503, 511, 544 and 550, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increased photostability and/or increased nanoparticletolerance relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes a mutation at position 370. In some embodiments, the mutationis selected from the group consisting of: H370G, H370T, H370S, H370K,H370R, H370A, H370Q, H370W, H370Y and H370F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. Optionally, thismodified polymerase comprises the amino acid mutation H370R. Typically,this modified polymerase can exhibit an increased branching ratio and/orincreased nucleotide binding affinity relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes a mutation at position 365, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: T365H, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W and T365Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationT365F. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes a mutation at position 372, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K372G, K372E, K372T,K372S, K372R, K372A, K372Q, K372W, K372Y and K372F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK372Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes a mutation at position 481, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: A481E, A481F, A481G,A481S, A481R, A481K, A481A, A481T, A481Q, A481W and A481Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationA481E. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes a mutation at position 509, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K509E, K509F, K509G,K509S, K509R, K509K, K509A, K509T, K509Q, K509W and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK509Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes an amino acid mutation at any one, two, three or more positionsselected from the group consisting of: 9, 12, 14, 15, 58, 59, 61, 63,98, 129, 176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256,300, 302, 310, 357, 360, 362, 365, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509,511, 526, 528, 529, 531, 535, 544, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this modified polymerase comprises the aminoacid substitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furthercomprises an amino acid mutation at any one, two, three or morepositions selected from the group consisting of: 2, 9, 12, 14, 15, 58,59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208,221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359,360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430,455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528,529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this modified polymerase comprises the aminoacid substitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio, increased nucleotide binding affinity, increasedphotostability and/or increased nanoparticle tolerance relative to areference polymerase having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, F383L, D384N, A481E, A481F, A481G,A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G,D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F,K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q,K509W, K509Y and K509F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6. In some embodiments, the modifiedpolymerase comprises the amino acid mutations Typically, this modifiedpolymerase can exhibit an increased branching ratio and/or increasednucleotide binding affinity relative to a reference polymerase havingthe amino acid sequence of SEQ ID NO: 7. Optionally, the modifiedpolymerase can further include one or more mutations reducing 3′ to 5′exonuclease activity selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at positions 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid substitutions E372Y and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at positions 365, 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid substitutions T365F, E372Y and K509Y, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at positions 365, 372, 481 and 509,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid substitutions T365F, E372Y, A481E and K509Y,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. Typically, such modified polymerases can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 7.Optionally, the modified polymerase can further include one or moremutations reducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furthercomprises the amino acid mutation H370R. Optionally, the polymerase canfurther comprise any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, R107K, T147A, K221R,V318A, L339M, D359E, E372K, D405E, V503A, K511I, A544R, M550T, E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F,K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 7. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increase t⁻¹ value inthe presence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the t⁻¹ value of the modified polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. Optionally, the modifiedpolymerase can further include one or more mutations reducing 3′ to 5′exonuclease activity selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include any one,two, three or more amino acid mutations selected from the groupconsisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, H370R,E372K, D405E, V503A, K511I, A544R and M550T, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. In someembodiments, the t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes the amino acid mutationH370R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increased t_(po1)value in the presence of the dye-labeled nucleotides relative to areference polymerase having the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the t_(po1) value of the modified polymerase isincreased by at least about 105%, 110%, 125%, 150%, 175%, 200%, 250%,500%, 750%, or 1000% relative to the reference polymerase. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. Optionally, the modified polymerase canfurther include any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, T147A, K221R, V318A,L339M, D359E, H370R, E372K, D405E, V503A, K511I, A544R and M550T,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the t_(po1) value is increased in thepresence of the dye-labeled nucleotide AF647-C6-dG6P. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid mutation K380R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furthercomprises any one, two, three or more amino acid mutations selected fromthe group consisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E,H370R, E372K, D405E, V503A, K511I, A544R and M550T, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, the modified polymerase can further comprise one, two orthree amino acid mutations selected from the group: T365G, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H, H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H,E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, A481E, A481F,A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 7. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 1 and furtherincludes an amino acid mutation selected from the group: T373G, T373E,T373T, T373S, T373R, T373A, K T373Q, T373W, T373Y and T373F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 1.Typically, this modified polymerase can exhibit an increased t⁻¹ valuein the presence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the t⁻¹ value of the modified polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. Optionally, the modifiedpolymerase can further include one or more mutations reducing 3′ to 5′exonuclease activity selected from the group consisting of: D12A, E14I,E14A, T15I, N62D, D66A, Y165F, Y165C, and D169A, wherein the numberingis relative to the amino acid sequence of SEQ ID NO: 1. In someembodiments, the t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes the amino acid mutationT373R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 1. In some embodiments, the modified polymerase furtherincludes the mutations D12A and D66A.

Also disclosed herein are exemplary nucleotide sequences(polynucleotides) encoding the modified polymerases of the presentdisclosure.

In some embodiments, the nucleotide sequence encodes a modifiedpolymerase having or comprising an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 7.

In some embodiments, the nucleotide sequence encoding the modifiedpolymerase having the amino acid sequence of SEQ ID NO: 7 comprises thefollowing nucleotide sequence:

(SEQ ID NO: 9) atgcctcgcaaaatgtttagctgcgattttgaaaccaccaccaaactggatgattgtcgtgtttgggcctatggctatatggaaattggcaacctggataattataaaatcggcaatagcctggatgaatttatgcagtgggttatggaaattcaggccgatctgtattttcataacctgaaatttgatggtgcctttattgtgaattggctggaacatcatggctttaaatggtctaatgaaggcctgccgaatacctataacaccatcattagcaaaatgggccagtggtatatgattgatatttgctttggctataaaggcaaacgtaaactgcataccgtgatttatgatagcctgaaaaaactgccgtttccggtgaaaaaaatcgccaaagatttccaattacctttactgaagggtgatattgattatcatgcagaacgtccggttggtcatgaaattacaccggaagaatatgaatacatcaagaatgatattgaaattattgcccgtgccctggatattcagtttaaacagggtctggatcgtatgaccgcaggtagcgattctctgaaaggctttaaagatattctgagcaccaaaaaatttaacaaagtgtttccgaaactgagcctgccgatggataaagaaattcgtcgtgcctatcgtggtggttttacctggctgaatgataaatataaagaaaaagaaattggcgaaggcatggtttttgatgttaatagcctgtatccgagccagatgtatagccgtccgctgccgtatggtgcaccgattgtgtttcagggcaaatatgaaaaagatgaacagtatccgctgtatattcagcgcatccgctttgaatttgaactgaaagaaggctatatcccgaccatccagattaaaaaaaatccgttttttaaaggcaacgaatatctgaaaaatagcggtgcagaaccggttgaactgtatctgaccaatgtggatctggaactgatccaggaacattatgaaatgtacaacgtggaatatattgatggttttaaatttcgcgaaaaaaccggtctgtttaaagagttcattgataaatggacctatgtgaaaacccatgaaaaaggtgcaaaaaaacagctggccaaactgatgttgaattccctgtatggcaaatttgcaagcaatccggatgttaccggtaaagttccgtatctgaaagaagatggtagcctgggttttcgtgttggtgatgaagaatataaagatccggtttataccccgatgggtgtttttattaccgcatgggcacgttttaccaccattaccgcagcacaggcatgttatgaccgcattatttattgcgataccgatagcattcatctgaccggcaccgaagttccggaaattattaaagatattgttgatccgaaaaaactgggttattgggcacatgaaagcacctttaaacgtgcaaaatatctgcgccagaaaacctatattcaggatatttatgccaaagaagtggacggtaaactgattgaatgttctccggatgaagcaaccaccacaaaatttagcgttaaatgcgcaggtatgaccgataccattaaaaaaaaagtgacctttgataactttcgtgtgggttttagcagcaccggtaaaccgaaaccggttcaggttaatggtggtgttgttctggttgatag cgtgtttaccattaaataa

In some embodiments, the nucleotide sequence of the modified polymerasecomprises a polynucleotide that is at least 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the nucleotide sequence of SEQ ID NO: 9.

In some embodiments, the modified polymerase comprising the amino acidsequence of SEQ ID NO: 7 can be encoded by the following nucleotidesequence:

(SEQ ID NO: 10) ATGCCTAGAAAAATGTTTAGTTGTGACTTTGAGACGACTACAAAGTTAGACGATTGTCGTGTATGGGCATATGGCTATATGGAAATCGGTAATCTCGACAACTACAAGATTGGAAATAGCTTAGATGAATTCATGCAGTGGGTTATGGAAATTCAAGCTGATTTATATTTCCACAATCTAAAATTTGACGGTGCTTTCATTGTAAACTGGTTAGAGCATCATGGTTTTAAGTGGTCAAACGAAGGGTTACCGAATACTTATAACACAATAATATCAAAAATGGGTCAATGGTATATGATTGACATATGTTTCGGCTATAAGGGAAAACGGAAATTACATACAGTGATATACGACAGCTTAAAGAAATTGCCGTTCCCAGTAAAGAAAATAGCGAAAGATTTTCAATTACCGTTATTGAAGGGTGACATTGATTACCACGCTGAACGTCCTGTTGGACATGAGATAACACCCGAAGAATACGAGTATATTAAGAACGACATAGAAATTATCGCACGTGCACTTGACATTCAATTTAAACAGGGTTTAGACCGAATGACAGCTGGGAGCGATAGCCTTAAAGGGTTTAAGGACATACTTAGCACCAAGAAATTTAACAAGGTGTTTCCTAAGCTTAGCCTACCAATGGATAAAGAAATAAGGCGAGCTTATCGTGGTGGCTTCACATGGTTAAACGATAAATACAAAGAAAAAGAGATTGGTGAAGGTATGGTGTTTGACGTTAACAGCCTATACCCCAGTCAGATGTATTCCCGACCACTCCCGTACGGAGCGCCAATCGTATTCCAAGGAAAGTATGAGAAAGATGAGCAATATCCGCTCTATATACAGCGTATCAGATTTGAGTTTGAATTGAAAGAGGGCTATATACCCACAATTCAGATTAAGAAAAATCCCTTTTTTAAGGGTAATGAGTATCTTAAAAACAGTGGCGCTGAGCCTGTTGAACTATATCTTACTAATGTAGATTTAGAATTAATACAGGAACACTACGAAATGTATAACGTTGAGTATATTGACGGATTTAAATTCCGTGAAAAGACTGGATTATTCAAAGAGTTTATTGATAAATGGACATATGTAAAAACTCATGAAAAGGGAGCTAAGAAACAATTGGCTAAGCTAATGTTGAATAGTCTCTATGGTAAATTTGCAAGTAACCCTGACGTTACAGGTAAAGTCCCTTATTTAAAAGAAGATGGGAGCCTTGGTTTCCGTGTTGGTGATGAGGAATATAAAGACCCTGTTTATACACCTATGGGTGTGTTTATAACGGCATGGGCTAGATTTACAACTATAACAGCGGCACAAGCGTGTTACGATAGAATTATATATTGTGACACTGATAGTATACATTTAACAGGTACAGAAGTACCAGAAATAATAAAGGATATTGTTGATCCAAAAAAGTTAGGGTACTGGGCGCATGAAAGCACATTTAAGAGAGCAAAATATTTACGTCAGAAAACGTATATTCAAGACATATATGCGAAAGAGGTTGACGGTAAATTGATAGAGTGTTCACCTGATGAAGCTACGACAACTAAATTCAGTGTGAAATGTGCCGGAATGACTGACACTATCAAAAAGAAAGTCACATTTGATAACTTTAGAGTTGGTTTCAGTAGCACGGGTAAACCTAAACCAGTTCAAGTTAATGGCGGGGTAGTGTTGGTTGATAG TGTGTTTACGATTAAA

In some embodiments, the nucleotide sequence of the modified polymerasecomprises a polynucleotide that is at least 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the nucleotide sequence of SEQ ID NO: 10.

In some embodiments, the polynucleotide encodes a modified polymerasehaving or comprising an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 7.

In some embodiments, the modified polymerase comprising the amino acidsequence of SEQ ID NO: 8 can be encoded by the following nucleotidesequence:

(SEQ ID NO: 11) atgcctcgcaaaatgtttagctgcgattttgaaaccaccaccaaactggatgattgtcgtgtttgggcctatggctatatggaaattggcaacctggataattataaaatcggcaatagcctggatgaatttatgcagtgggttatggaaattcaggccgatctgtattttcataacctgaaatttgatggtgcctttattgtgaattggctggaacatcatggctttaaatggtctaatgaaggcctgccgaatacctataacaccatcattagcaaaatgggccagtggtatatgattgatatttgctttggctataaaggcaaacgtaaactgcataccgtgatttatgatagcctgaaaaaactgccgtttccggtgaaaaaaatcgccaaagatttccaattacctttactgaagggtgatattgattatcatgcagaacgtccggttggtcatgaaattacaccggaagaatatgaatacatcaagaatgctattgaaattattgcccgtgccctggatattcagtttaaacagggtctggatcgtatgaccgcaggtagcgattctctgaaaggctttaaagatattctgagcaccaaaaaatttaacaaagtgtttccgaaactgagcctgccgatggataaagaaattcgtcgtgcctatcgtggtggttttacctggctgaatgataaatataaagaaaaagaaattggcgaaggcatggtttttgatgttaatagcctgtatccgagccagatgtatagccgtccgctgccgtatggtgcaccgattgtgtttcagggcaaatatgaaaaagatgaacagtatccgctgtatattcagcgcatccgctttgaatttgaactgaaagaaggctatatcccgaccatccagattaaaaaaaatccgttttttaaaggcaacgaatatctgaaaaatagcggtgcagaaccggttgaactgtatctgaccaatgtggatctggaactgatccaggaacattatgaaatgtacaacgtggaatatattgatggttttaaatttcgcgaaaaaaccggtctgtttaaagagttcattgataaatggacctatgtgaaaacccatgaaaaaggtgcaaaaaaacagctggccaaactgatgttgaattccctgtatggcaaatttgcaagcaatccggatgttaccggtaaagttccgtatctgaaagaagatggtagcctgggttttcgtgttggtgatgaagaatataaagatccggtttataccccgatgggtgtttttattaccgcatgggcacgttttaccaccattaccgcagcacaggcatgttatgaccgcattatttattgcgataccgatagcattcatctgaccggcaccgaagttccggaaattattaaagatattgttgatccgaaaaaactgggttattgggcacatgaaagcacctttaaacgtgcaaaatatctgcgccagaaaacctatattcaggatatttatgccaaagaagtggacggtaaactgattgaatgttctccggatgaagcaaccaccacaaaatttagcgttaaatgcgcaggtatgaccgataccattaaaaaaaaagtgacctttgataactttcgtgtgggttttagcagcaccggtaaaccgaaaccggttcaggttaatggtggtgttgttctggttgatag cgtgtttaccattaaataa

In some embodiments, the nucleotide sequence of the modified polymerasecomprises a polynucleotide that is at least 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the nucleotide sequence of SEQ ID NO: 11.

In some embodiments, the modified polymerase comprising the amino acidsequence of SEQ ID NO: 8 can be encoded by the following nucleotidesequence:

(SEQ ID NO: 12) ATGCCTAGAAAAATGTTTAGTTGTGACTTTGAGACGACTACAAAGTTAGACGATTGTCGTGTATGGGCATATGGCTATATGGAAATCGGTAATCTCGACAACTACAAGATTGGAAATAGCTTAGATGAATTCATGCAGTGGGTTATGGAAATTCAAGCTGATTTATATTTCCACAATCTAAAATTTGACGGTGCTTTCATTGTAAACTGGTTAGAGCATCATGGTTTTAAGTGGTCAAACGAAGGGTTACCGAATACTTATAACACAATAATATCAAAAATGGGTCAATGGTATATGATTGACATATGTTTCGGCTATAAGGGAAAACGGAAATTACATACAGTGATATACGACAGCTTAAAGAAATTGCCGTTCCCAGTAAAGAAAATAGCGAAAGATTTTCAATTACCGTTATTGAAGGGTGACATTGATTACCACGCTGAACGTCCTGTTGGACATGAGATAACACCCGAAGAATACGAGTATATTAAGAACGCCATAGAAATTATCGCACGTGCACTTGACATTCAATTTAAACAGGGTTTAGACCGAATGACAGCTGGGAGCGATAGCCTTAAAGGGTTTAAGGACATACTTAGCACCAAGAAATTTAACAAGGTGTTTCCTAAGCTTAGCCTACCAATGGATAAAGAAATAAGGCGAGCTTATCGTGGTGGCTTCACATGGTTAAACGATAAATACAAAGAAAAAGAGATTGGTGAAGGTATGGTGTTTGACGTTAACAGCCTATACCCCAGTCAGATGTATTCCCGACCACTCCCGTACGGAGCGCCAATCGTATTCCAAGGAAAGTATGAGAAAGATGAGCAATATCCGCTCTATATACAGCGTATCAGATTTGAGTTTGAATTGAAAGAGGGCTATATACCCACAATTCAGATTAAGAAAAATCCCTTTTTTAAGGGTAATGAGTATCTTAAAAACAGTGGCGCTGAGCCTGTTGAACTATATCTTACTAATGTAGATTTAGAATTAATACAGGAACACTACGAAATGTATAACGTTGAGTATATTGACGGATTTAAATTCCGTGAAAAGACTGGATTATTCAAAGAGTTTATTGATAAATGGACATATGTAAAAACTCATGAAAAGGGAGCTAAGAAACAATTGGCTAAGCTAATGTTGAATAGTCTCTATGGTAAATTTGCAAGTAACCCTGACGTTACAGGTAAAGTCCCTTATTTAAAAGAAGATGGGAGCCTTGGTTTCCGTGTTGGTGATGAGGAATATAAAGACCCTGTTTATACACCTATGGGTGTGTTTATAACGGCATGGGCTAGATTTACAACTATAACAGCGGCACAAGCGTGTTACGATAGAATTATATATTGTGACACTGATAGTATACATTTAACAGGTACAGAAGTACCAGAAATAATAAAGGATATTGTTGATCCAAAAAAGTTAGGGTACTGGGCGCATGAAAGCACATTTAAGAGAGCAAAATATTTACGTCAGAAAACGTATATTCAAGACATATATGCGAAAGAGGTTGACGGTAAATTGATAGAGTGTTCACCTGATGAAGCTACGACAACTAAATTCAGTGTGAAATGTGCCGGAATGACTGACACTATCAAAAAGAAAGTCACATTTGATAACTTTAGAGTTGGTTTCAGTAGCACGGGTAAACCTAAACCAGTTCAAGTTAATGGCGGGGTAGTGTTGGTTGATAG TGTGTTTACGATTAAA

In some embodiments, the nucleotide sequence of the modified polymerasecomprises a polynucleotide that is at least 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the nucleotide sequence of SEQ ID NO: 12.

In some embodiments, the polynucleotide encodes a modified polymerasehaving or comprising an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 8, wherein the modified polymerase comprises any residue other thanaspartic acid (D) at position 166, the numbering being relative to theamino acid sequence of SEQ ID NO: 8. In some embodiments, the residue atposition 166 is alanine (A).

In some embodiments, the polynucleotide encodes a modified polymerasehaving or comprising an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 8.

As the skilled artisan will appreciate, due to the degeneracy of thegenetic code, many different polynucleotides can encode the modifiedpolymerases disclosed herein, all of which are within the scope of thepresent disclosure. An exemplary list of the various nucleotide “codons”or “triplets” that may encode a given amino acid is provided in Table 1,below:

TABLE 1 Amino Acid SLC DNA codons Isoleucine I ATT, ATC, ATA Leucine LCTT, CTC, CTA, CTG, TTA, TTG Valine V GTT, GTC, GTA, GTG Phenylalanine FTTT, TTC Methionine M ATG Cysteine C TGT, TGC Alanine A GCT, GCC, GCA,GCG Glycine G GGT, GGC, GGA, GGG Proline P CCT, CCC, CCA, CCG ThreonineT ACT, ACC, ACA, ACG Serine S TCT, TCC, TCA, TCG, AGT, AGC Tyrosine YTAT, TAC Tryptophan W TGG Glutamine Q CAA, CAG Asparagine N AAT, AACHistidine H CAT, CAC Glutamic acid E GAA, GAG Aspartic acid D GAT, GACLysine K AAA, AAG Arginine R CGT, CGC, CGA, CGG, AGA, AGG Stop codonsStop TAA, TAG, TGA

Table 1 lists the twenty naturally occurring amino acids commonly foundin proteins, along with the single-letter code (“SLC”) typically used torepresent these amino acids in protein databases. The nucleotide codonsthat typically encode each amino acid are also listed. As shown in Table1, 3 of the 64 possible 3-letter combinations of the nucleic acid codingunits T, C, A and G can be used to encode one of the three stop codons(TAA, TAG and TGA) that typically signals the end of a sequence. Theremaining 61 combinations can be used to encode one of the twentynaturally-occurring amino acids, as indicated in Table 1. As the skilledartisan will readily appreciate, the corresponding peptide sequenceencoded by any given polynucleotide sequence can typically be determinedunambiguously. Because most amino acids have multiple codons, however, anumber of different polynucleotide sequences can encode the same proteinsequence.

As indicated in Table 1, most of the naturally occurring amino acids arecoded for by multiple codons. Because codon preference can vary fromspecies to species, selection or synthesis of a polynucleotide sequence,and its expression in a given species can optionally be optimized byselecting for codons that are highly preferred, or provide optimaltranslation efficiency or overall expression levels, in the species ofchoice. See, for example, Kudla et al., Science 324:255-258 (2009). Insome embodiments the polynucleotide may be homologous to polynucleotideshaving the nucleotide sequence of SEQ ID NO: 9, 10, 11 or 12, andcontain “silent” or “synonymous” mutations that do not alter the peptidesequence of the encoded peptide product.

In some embodiments, the polynucleotide is an artificial or recombinantnucleic acid molecule that hybridizes to a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO: 12 under highly stringent conditions over substantially theentire length of the nucleic acid molecule. Hybridization, orassociation of nucleic acid molecules due to hydrogen bonding, basestacking, solvent exclusion and the like are well known in the art andhave been extensively described. See, e.g., Hanes et al., Gene Probes(Oxford University Press 1995); Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology (Elsevier, 1993). In one exemplarymethod, the polynucleotide is greater than 100 nucleotides in length andis subjected to high-stringency hybridization conditions comprising 50%formalin with 1 mg of heparin at 42° C. overnight. In some embodiments,the polynucleotide can also be subjected to a high-stringency washfollowing hybridization. In some embodiments, the high stringency washcan comprise one wash with 0.2×.SSC at 65° C. for 15 minutes. In someembodiments, the polynucleotide can be subjected to extremely stringentconditions selected to be equal to the thermal melting point (T_(m)) fora particular polynucleotide sequence. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of the test sequencehybridizes to a perfectly matched probe. For the purposes of the presentinvention, generally, “highly stringent” hybridization and washconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence at a defined ionic strength and pH.

In some embodiments, disclosed herein is a polynucleotide encodingmodified polymerases, wherein the polynucleotide specifically hybridizesto a probe nucleic acid having the nucleotide sequence of SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 under stringent, highlystringent or extremely stringent conditions. A polynucleotide is said tospecifically hybridize to a probe nucleic acid when it hybridizes atleast 50% as well to the probe as to the perfectly matched complementarytarget, i.e., with a signal to noise ratio at least half as high ashybridization of the probe to the target under conditions in which theperfectly matched probe binds to the perfectly matched complementarytarget with a signal to noise ratio that is at least about 5-10 times ashigh as that observed for hybridization to any of the unmatched targetnucleic acids.

The skilled artisan will also appreciate that many variants of thedisclosed nucleotide and/or amino acid sequences are within the scopeand spirit of the present disclosure. For example, modified polymerasescomprising one or more conservative mutations of the disclosed aminoacid sequences and are functionally similar to the modified polymerasesexplicitly disclosed herein are also disclosed. In some embodiments, themodified polymerases of the present disclosure can be a variant of thevarious modified polymerases disclosed herein and additionally containone or more conservative mutations.

In some embodiments, the modified polymerase is a variant of apolymerase comprising the amino acid sequence of SEQ ID NO: 7, whereinthe variant polymerase further comprises one or more amino acidsubstitutions within one or more amino acid motifs selected from thegroup consisting of: a motif comprising the amino acid sequence of SEQID NO: 2; a motif comprising the amino acid sequence of SEQ ID NO: 3; amotif comprising the amino acid sequence of SEQ ID NO: 4; and a motifcomprising the amino acid sequence of SEQ ID NO: 5. In some embodiments,the one or more substitutions are conservative mutations. In someembodiments, the one or more substitutions are non-conservativemutations.

In some embodiments, the modified polymerase is derived from a M2polymerase (also known as M2Y DNA polymerase) having the amino acidsequence of the SEQ ID NO: 13 as follows:

(SEQ ID NO: 13) 1msrkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl 61kfdgafivnw leqhgfkwsn eglpntynti iskmgqwymi dicfgykgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhterp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir kayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgvep velyltnvdl eliqehyely nveyidgfkf rektglfkdf 361idkwtyvkth eegakkqlak lmlnslygkf asnpdvtgkv pylkddgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyvkevdgkl kecspdeatt tkfsvkcagm tdtikkkvtf 541dnfavgfssm gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% or 100%identical to the amino acid of SEQ ID NO: 13. In some embodiments, themodified polymerase can have a nanoparticle tolerance that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000 relative to the nanoparticletolerance of a reference Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the modified polymerasehas a photostability that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000 relative to the photostability of a reference Phi-29 polymerasehaving the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 13 and further comprises an aminoacid mutation at one, two, three or more amino acid positions selectedfrom the group consisting of: 9, 11, 12, 58, 59, 63, 162, 162, 166, 377and 385, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. Typically, such a modified polymerase will exhibit reduced3′ to 5′ exonuclease activity relative to reference polymerase havingthe amino acid sequence of SEQ ID NO:13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 13 and further comprises one, two,three or more amino acid mutations selected from the group consistingof: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377Aand S385G, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 6. In some embodiments, the modified polymerase comprisesany one, two, three, four, five or all of these mutations. In someembodiments, the modified polymerase comprises the amino acidsubstitution D166A. In some embodiments, the modified polymerasecomprises the amino acid substitutions D9A and D63A. In someembodiments, the modified polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, such modified polymerases willexhibit reduced 3′ to 5′ exonuclease activity relative to referencepolymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furthercomprises an amino acid substitution at one or more positions selectedfrom the group consisting of: 2, 73, 147, 221, 318, 339, 359, 372, 405,503, 511, 544 and 550, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6. Typically, this modified polymerase canexhibit increased photostability and/or increased nanoparticle tolerancerelative to a reference polymerase having the amino acid sequence of SEQID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes a mutation at position 370. In some embodiments, the mutationis selected from the group consisting of: H370G, H370T, H370S, H370K,H370R, H370A, H370Q, H370W, H370Y and H370F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. Optionally, thismodified polymerase comprises the amino acid mutation H370R. Typically,this modified polymerase can exhibit an increased branching ratio and/orincreased nucleotide binding affinity relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes a mutation at position 365, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: T365H, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W and T365Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationT365F. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes a mutation at position 372, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K372G, K372E, K372T,K372S, K372R, K372A, K372Q, K372W, K372Y and K372F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK372Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes a mutation at position 481, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: A481E, A481F, A481G,A481S, A481R, A481K, A481A, A481T, A481Q, A481W and A481Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationA481E. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes a mutation at position 509, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K509E, K509F, K509G,K509S, K509R, K509K, K509A, K509T, K509Q, K509W and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK509Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E,E372K, D405E, V503A, K511I, A544R and M550T, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. Typically, thismodified polymerase can exhibit increased photostability and/orincreased nanoparticle tolerance relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes an amino acid mutation at any one, two, three or more positionsselected from the group consisting of: 9, 12, 14, 15, 58, 59, 61, 63,98, 129, 176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256,300, 302, 310, 357, 360, 362, 365, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509,511, 526, 528, 529, 531, 535, 544, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this modified polymerase comprises the aminoacid substitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furthercomprise amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248,251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385,386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481,483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544,550, 552, 555, 567, 569 and 572, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 6. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions.Optionally, this modified polymerase comprises the amino acidsubstitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio, increased nucleotide binding affinity, increasedphotostability and/or increased nanoparticle tolerance relative to areference polymerase having the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. In some embodiments, the modified polymerase comprises theamino acid mutations Typically, this modified polymerase can exhibit anincreased branching ratio and/or increased nucleotide binding affinityrelative to a reference polymerase having the amino acid sequence of SEQID NO: 13. Optionally, the modified polymerase can further include oneor more mutations reducing 3′ to 5′ exonuclease activity selected fromthe group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes amino acid mutations at positions 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes the amino acid substitutions E372Y and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes amino acid mutations at positions 365, 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes the amino acid substitutions T365F, E372Y and K509Y, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes amino acid mutations at positions 365, 372, 481 and 509,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes the amino acid substitutions T365F, E372Y, A481E and K509Y,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. Typically, such modified polymerases can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 13.Optionally, the modified polymerase can further include one or moremutations reducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furthercomprises the amino acid mutation H370R. Optionally, the polymerase canfurther comprise any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, R107K, T147A, K221R,V318A, L339M, D359E, E372K, D405E, V503A, K511I, A544R, M550T, E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F,K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 13. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furthercomprises any one, two, three or more amino acid mutations selected fromthe group consisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E,H370R, E372K, D405E, V503A, K511I, A544R and M550T, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, the modified polymerase can further comprise one, two orthree amino acid mutations selected from the group: T365G, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H, H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H,E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, A481E, A481F,A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 13. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increase t⁻¹ value inthe presence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 13. In someembodiments, the t⁻¹ value of the modified polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. Optionally, the modifiedpolymerase can further include one or more mutations reducing 3′ to 5′exonuclease activity selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include any one,two, three or more amino acid mutations selected from the groupconsisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, H370R,E372K, D405E, V503A, K511I, A544R and M550T, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes the amino acid mutationH370R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 13 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increased t_(po1)value in the presence of the dye-labeled nucleotides relative to areference polymerase having the amino acid sequence of SEQ ID NO: 13. Insome embodiments, the t_(po1) value of the modified polymerase isincreased by at least about 105%, 110%, 125%, 150%, 175%, 200%, 250%,500%, 750%, or 1000% relative to the reference polymerase. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. Optionally, the modified polymerase canfurther include any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, T147A, K221R, V318A,L339M, D359E, H370R, E372K, D405E, V503A, K511I, A544R and M550T,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. In some embodiments, the t_(po1) value is increased in thepresence of the dye-labeled nucleotide AF647-C6-dG6P. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid mutation K380R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase is derived from abacteriophage Nf polymerase having the amino acid sequence of the SEQ IDNO: 14 as follows:

(SEQ ID NO: 14) 1msrkmfscdf etttklddcr vwaygymeig nldnykigns ldefmqwvme iqadlyfhnl 61kfdgafivnw leqhgfkwsn eglpntynti iskmgqwymi dicfgyrgkr klhtviydsl 121kklpfpvkki akdfqlpllk gdidyhterp vgheitpeey eyikndieii araldiqfkq 181gldrmtagsd slkgfkdils tkkfnkvfpk lslpmdkeir kayrggftwl ndkykekeig 241egmvfdvnsl ypsqmysrpl pygapivfqg kyekdeqypl yiqrirfefe lkegyiptiq 301ikknpffkgn eylknsgvep velyltnvdl eliqehyely nveyidgfkf rektglfkdf 361idkwtyvkth eegakkqlak lmlnslygkf asnpdvtgkv pylkddgslg frvgdeeykd 421pvytpmgvfi tawarfttit aaqacydrii ycdtdsihlt gtevpeiikd ivdpkklgyw 481ahestfkrak ylrqktyiqd iyvkevdgkl kecspdeatt tkfsvkcagm tdtikkkvtf 541dnfavgfssm gkpkpvqvng gvvlvdsvft ik

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% or 100%identical to the amino acid of SEQ ID NO: 14. In some embodiments, themodified polymerase can have a nanoparticle tolerance that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000 relative to the nanoparticletolerance of a reference Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the modified polymerasehas a photostability that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000 relative to the photostability of a reference Phi-29 polymerasehaving the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the modified Nf polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 14 and further comprises an aminoacid mutation at one, two, three or more amino acid positions selectedfrom the group consisting of: 9, 11, 12, 58, 59, 63, 162, 162, 166, 377and 385, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. Typically, such a modified polymerase will exhibit reduced3′ to 5′ exonuclease activity relative to reference polymerase havingthe amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 14 and further comprises one, two,three or more amino acid mutations selected from the group consistingof: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377Aand S385G, wherein the numbering is relative to the amino acid sequenceof SEQ ID NO: 6. In some embodiments, the modified polymerase comprisesany one, two, three, four, five or all of these mutations. In someembodiments, the modified polymerase comprises the amino acidsubstitution D166A. In some embodiments, the modified polymerasecomprises the amino acid substitutions D9A and D63A. In someembodiments, the modified polymerase comprises the amino acidsubstitutions N59D and T12I. Typically, such modified polymerases willexhibit reduced 3′ to 5′ exonuclease activity relative to referencepolymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furthercomprises an amino acid substitution at one or more positions selectedfrom the group consisting of: 2, 73, 107, 147, 221, 318, 339, 359, 372,405, 503, 511, 544 and 550, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increased photostability and/or increased nanoparticletolerance relative to a reference polymerase having the amino acidsequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes a mutation at position 370. In some embodiments, the mutationis selected from the group consisting of: H370G, H370T, H370S, H370K,H370R, H370A, H370Q, H370W, H370Y and H370F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. Optionally, thismodified polymerase comprises the amino acid mutation H370R. Typically,this modified polymerase can exhibit an increased branching ratio and/orincreased nucleotide binding affinity relative to a reference polymerasehaving the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes a mutation at position 365, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: T365H, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W and T365Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationT365F. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes a mutation at position 372, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K372G, K372E, K372T,K372S, K372R, K372A, K372Q, K372W, K372Y and K372F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK372Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes a mutation at position 481, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: A481E, A481F, A481G,A481S, A481R, A481K, A481A, A481T, A481Q, A481W and A481Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationA481E. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes a mutation at position 509, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themutation is selected from the group consisting of: K509E, K509F, K509G,K509S, K509R, K509K, K509A, K509T, K509Q, K509W and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, this modified polymerase comprises the amino acid mutationK509Y. Typically, this modified polymerase can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: S2P, Q73H, R107K, T147A, K221R, V318A, L339M,D359E, E372K, D405E, V503A, K511I, A544R and M550T, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Typically, this modified polymerase can exhibit increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes an amino acid mutation at any one, two, three or more positionsselected from the group consisting of: 9, 12, 14, 15, 58, 59, 61, 63,98, 129, 176, 185, 186, 187, 195, 208, 246, 247, 248, 251, 252, 256,300, 302, 310, 357, 360, 362, 365, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 507, 509,511, 526, 528, 529, 531, 535, 544, 555, 567, 569 and 572, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. Optionally, this modified polymerase comprises the aminoacid substitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furthercomprise amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions.Optionally, this modified polymerase comprises the amino acidsubstitution H370R. In some embodiments, the polymerase furthercomprises one or more mutations reducing the exonuclease activity asdescribed herein such as, for example, the amino acid substitutionD166A. Typically, this modified polymerase can exhibit increasedbranching ratio, increased nucleotide binding affinity, increasedphotostability and/or increased nanoparticle tolerance relative to areference polymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 6. In some embodiments, the modified polymerase comprises theamino acid mutations Typically, this modified polymerase can exhibit anincreased branching ratio and/or increased nucleotide binding affinityrelative to a reference polymerase having the amino acid sequence of SEQID NO: 14. Optionally, the modified polymerase can further include oneor more mutations reducing 3′ to 5′ exonuclease activity selected fromthe group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes amino acid mutations at positions 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes the amino acid substitutions E372Y and K509Y, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes amino acid mutations at positions 365, 372 and 509, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes the amino acid substitutions T365F, E372Y and K509Y, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 6. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes amino acid mutations at positions 365, 372, 481 and 509,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes the amino acid substitutions T365F, E372Y, A481E and K509Y,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. Typically, such modified polymerases can exhibit an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference polymerase having the amino acid sequence of SEQ ID NO: 14.Optionally, the modified polymerase can further include one or moremutations reducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furthercomprises the amino acid mutation H370R. Optionally, the polymerase canfurther comprise any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, R107K, T147A, K221R,V318A, L339M, D359E, E372K, D405E, V503A, K511I, A544R, M550T, E371G,E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F,K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes the amino acid mutation H370R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 6. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furthercomprises any one, two, three or more amino acid mutations selected fromthe group consisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E,H370R, E372K, D405E, V503A, K511I, A544R and M550T, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 6.Optionally, the modified polymerase can further comprise one, two orthree amino acid mutations selected from the group: T365G, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H, H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H,E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, A481E, A481F,A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO:6. Typically, this modified polymerasecan exhibit increase branching ratio and/or increased photostabilityand/or increased nanoparticle tolerance relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 14. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO:6.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes an amino acid mutation selected from the group: H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y and H370F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increase t⁻¹ value inthe presence of the dye-labeled nucleotides relative to a referencepolymerase having the amino acid sequence of SEQ ID NO: 14. In someembodiments, the t⁻¹ value of the modified polymerase is increased by atleast about 105%, 110%, 125%, 150%, 175%, 200%, 250%, 500%, 750%, or1000% relative to the reference polymerase. Optionally, the modifiedpolymerase can further include one or more mutations reducing 3′ to 5′exonuclease activity selected from the group consisting of: D9A, E11A,E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include any one,two, three or more amino acid mutations selected from the groupconsisting of: S2P, Q73H, T147A, K221R, V318A, L339M, D359E, H370R,E372K, D405E, V503A, K511I, A544R and M550T, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. In someembodiments, the t⁻¹ value is increased in the presence of thedye-labeled nucleotide AF647-C6-dG6P. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14 and further includes the amino acid mutationH370R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 14 and furtherincludes an amino acid mutation selected from the group: K380G, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y and K380F, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Typically, this modified polymerase can exhibit an increased t_(po1)value in the presence of the dye-labeled nucleotides relative to areference polymerase having the amino acid sequence of SEQ ID NO: 14. Insome embodiments, the t_(po1) value of the modified polymerase isincreased by at least about 105%, 110%, 125%, 150%, 175%, 200%, 250%,500%, 750%, or 1000% relative to the reference polymerase. Optionally,the modified polymerase can further include one or more mutationsreducing 3′ to 5′ exonuclease activity selected from the groupconsisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C,D166A, Q377A and S385G, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. Optionally, the modified polymerase canfurther include any one, two, three or more amino acid mutationsselected from the group consisting of: S2P, Q73H, T147A, K221R, V318A,L339M, D359E, H370R, E372K, D405E, V503A, K511I, A544R and M550T,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 6. In some embodiments, the t_(po1) value is increased in thepresence of the dye-labeled nucleotide AF647-C6-dG6P. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes the amino acid mutation K380R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the modified polymerase is derived from an RB69polymerase having the amino acid sequence of the SEQ ID NO: 15 asfollows:

(SEQ ID NO: 15) MKEFYLTVEQIGDSIFERYIDSNGRERTREVEYKPSLFAHCPESQATKYFDIYGKPCTRKLFANMRDASQWIKRMEDIGLEALGMDDFKLAYLSDTYNYEIKYDHTKIRVANFDIEVTSPDGFPEPSQAKHPIDAITHYDSIDDRFYVFDLLNSPYGNVEEWSIEIAAKLQEQGGDEVPSEIIDKIIYMPFDNEKELLMEYLNFWQQKTPVILTGWNVESFDIPYVYNRIKNIFGESTAKRLSPHRKTRVKVIENMYGSREIITLFGISVLDYIDLYKKFSFTNQPSYSLDYISEFELNVGKLKYDGPISKLRESNHQRYISYNIIDVYRVLQIDAKRQFINLSLDMGYYAKIQIQSVFSPIKTWDAIIFNSLKEQNKVIPQGRSHPVQPYPGAFVKEPIPNRYKYVMSFDLTSLYPSIIRQVNISPETIAGTFKVAPLHDYINAVAERPSDVYSCSPNGMMYYKDRDGVVPTEITKVFNQRKEHKGYMLAAQRNGEIIKEALHNPNLSVDEPLDVDYRFDFSDEIKEKIKKLSAKSLNEMLFRAQRTEVAGMTAQINRKLLINSLYGALGNVWFRYYDLRNATAITTFGQMALQWIERKVNEYLNEVCGTEGEAFVLYGDTDSIYVSADKIIDKVGESKFRDTNHWVDFLDKFARERMEPAIDRGFREMCEYMNNKQHLMFMDREAIAGPPLGSKGIGGFWTGKKRYALNVWDMEGTRYAEPKLKIMGLETQKSSTPKAVQKALKECIRRMLQEGEESLQEYFKEFEKEFRQLNYISIASVSSANNIAKYDVGGFPGPKCPFHIRGILTYNRAIKGNIDAPQVVEGEKVYVLPLREGNPFGDKCIAWPSGTEITDLIKDDVLHWMDYTVLLEKTFIKPLEGFTSAAKLDYEKKASLFDM FDF

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical tothe amino acid of SEQ ID NO: 15.

In some embodiments, the modified polymerases disclosed herein, as wellas the modified polymerases encoded by the polynucleotides of thepresent disclosure, can exhibit (or can be further engineered or evolvedto exhibit) an altered polymerase activity in a particular biologicalassay of interest as compared to a reference counterpart. Some examplesof evolved polymerases having altered functions or activities can befound, for example, in U.S. Provisional Application No. 61/020,995,filed Jan. 14, 2008; International PCT Application No. WO 2008/091847,published Jul. 23, 2009; U.S. Publication No. 2007/0196846 A1, publishedAug. 23, 2007; U.S. Publication No. 2008/0108082, published May 8, 2008;and U.S. Publication No. 2009/0176233, published Jul. 9, 2009.

In some embodiments, a vector comprises a polynucleotide encoding amodified polymerase of the present disclosure. In some embodiments, thevector is a cosmid, plasmid, virus, phage or the like. In someembodiments, the vector is an expression vector. In some embodiments,the modified polymerases of the present disclosure can be produced by asuitable expression vector/host cell system. For example, a modifiedpolymerase can be encoded by suitable recombinant expression vectorscarrying inserted sequences of the modified polymerase. The polymerasesequence can be linked to a suitable expression vector. The polymerasesequence can be inserted in-frame into the suitable expression vector.The suitable expression vector can replicate in a phage host, or aprokaryotic or eukaryotic host cell. The suitable expression vector canreplicate autonomously in the host cell, or can be inserted into thehost cell's genome and be replicated as part of the host genome. Thesuitable expression vector can carry a selectable marker that confersresistance to drugs (e.g., kanamycin, ampicillin, tetracycline,chloramphenicol, or the like) or a requirement for a nutrient. Thesuitable expression vector can have one or more restriction sites forinserting the nucleic acid molecule of interest. The suitable expressionvector can include expression control sequences for regulatingtranscription and/or translation of the encoded sequence. The expressioncontrol sequences can include: promoters (e.g., inducible orconstitutive), enhancers, transcription terminators, and secretionsignals. The expression vector can be a plasmid, cosmid, virus, phage,and the like. The expression vector can enter a host cell which canreplicate the vector, produce an RNA transcript of the insertedsequence, and/or produce protein encoded by the inserted sequence.Methods for preparing suitable recombinant expression vectors andexpressing the RNA and/or protein encoded by the inserted sequences arewell known (Sambrook et al, Molecular Cloning (1989)). In someembodiments, the polynucleotide comprises the nucleotide sequence of SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a nucleotidesequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical toany of these nucleotide sequences. In some embodiments, thepolynucleotide is cloned into the expression vector pTTQ, which allowsexpression of the polypeptide encoded by the polynucleotide from thepTAC promoter (Stark, J. J. R., Gene, 51, pp. 255-267, 1987).

In some embodiments, the modified polymerase can be homologous to anaturally occurring polymerase; alternatively, it can be a biologicallyactive fragment, mutant or other derivative of a naturally occurringpolymerase. Typically, the polymerase will elongate a pre-existingnucleic acid, for example, a primer, by polymerizing nucleotides on tothe 3′ end of the molecule; for example, the polymerase can catalyzetransfer of a nucleoside monophosphate from a nucleoside triphosphate(or analog thereof) to the 3′ hydroxyl group of the polymerizationinitiation site. The polymerase can bind a target nucleic acid molecule,which may or may not be base-paired with a polymerization initiationsite (e.g., primer). The polymerase can bind a nucleotide. Thepolymerase can mediate incorporation of a nucleotide (or analog thereof)on to a polymerization initiation site (e.g., terminal 3′OH of aprimer). The polymerase can mediate cleavage between the α and βphosphate groups. The polymerase can mediate phosphodiester bondformation. The polymerase can mediate release of the polyphosphateproduct.

In some embodiments, the modified polymerase can be linked to a label toform a labeled polymerase that retains polymerase activity. In someembodiments, the label is an organic label. In some embodiments, thelabel is an inorganic label, for example a nanoparticle. Thenanoparticle can be a nanocrystal or a quantum dot. In some embodiments,the nanoparticle or quantum dot, or populations thereof, can comprise asurface layer including multiple functional groups such as, for example,dipeptides, tripeptides, monodentate thiols, or polydentate thiols aswell as mixtures of dipeptides, tripeptides, monodentate thiols, orpolydentate thiols with hydrophobic ligands like TDPA, OPA, TOP, and/orTOPO.

In some embodiments, the modified polymerase is linked to an energytransfer moiety. The energy transfer moiety can in some embodiments be aRET or FRET moiety. In some embodiments, the energy transfer moiety is adonor moiety. Exemplary methods of attaching labels to a polymerase canbe found, for example, in U.S. Provisional Application No. 61/184,770,filed Jun. 5, 2009.

In some embodiments, the polymerase can be further modified to compriseintact subunits, biologically-active fragments and/or fusion variants ofany polymerases disclosed herein, as well as mutated, truncated,modified, genetically engineered or fusion variants of such polymerases.The modifications of a modified polymerase can include amino acidsubstitutions, insertions, or deletions. In some embodiments, themodified polymerases can be isolated from a cell, or generated usingrecombinant DNA technology or chemical synthesis methods. In anotherembodiment, the modified polymerases can be expressed in prokaryote,eukaryote, viral, or phage organisms. In some embodiments, the modifiedpolymerases can be post-translationally modified proteins or fragmentsthereof.

The polymerases disclosed herein can also be derived from any subunits,mutated, modified, truncated, genetically engineered or fusion variantsof a polymerase having the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8 (wherein the mutation involves the replacement of one or more ormany amino acids with other amino acids, the insertion or deletion ofone or more or many amino acids, or the conjugation of parts of one ormore polymerases).

In some embodiments, the modified polymerase is a fusion proteincomprising some or all of the amino acid sequence of SEQ ID NO: 7 or SEQID NO: 8.

In some embodiments, the modified polymerase can be attached, fused orotherwise associated with a moiety that facilitates purification of thepolymerase, isolation of the polymerase and/or linkage of the polymeraseto one or more labels. For example, in some embodiments the moiety canbe an enzymatic recognition site, an epitope or an affinity tag thatfacilitates purification of the polymerase. In some embodiments, the tagis an affinity tag. The affinity tag can be selected from: a His tag, aGST tag, an HA tag, a SNAP-tag, and the like. In some embodiments, themodified polymerase can comprise a plurality of 6-His tags (SEQ ID NO:48), a plurality of GST tags, a plurality of HA tags, and combinationsthereof. Such tags can be inserted into any suitable position within thepolymerase sequence. In some embodiments, the modified polymerasecomprises one or more tags that are fused in-frame with the polymeraseamino acid sequence at the N-terminal, the C-terminal end, or anywherein between. Typically, the presence of the tag does not affectpolymerase activity.

In some embodiments, the affinity tag is a His tag. For example, in someembodiments the His tag can include a stretch of amino acids comprisingmultiple histidine residues. In some embodiments, the His tag comprisesa 6-His tag (SEQ ID NO: 48) (hexahistidine tag [SEQ ID NO: 48]). The Histag can optionally bind to one or more metal atom of a label. In oneexemplary embodiment, the His tag binds to bound metal ions on thesurface of a nanoparticle, for example, Ni²⁺, Co²⁺, or Cu²⁺ ions, thuslinking the polymerase to the nanoparticle. In some embodiments, the Histag comprises 2, 3, 4, 5, 6, 7, 8 or more histidine residues. In someembodiments, the His tag is fused to the N- or C-terminus of theprotein; alternatively, it can be fused at any suitable location withinthe open reading frame of the protein.

In some embodiments, the His tag may be fused directly with the protein;alternatively, a linker comprising various lengths of amino acidresidues can be placed between the protein and the His tag. The linkercan be flexible or rigid.

In some embodiments, the His tag can facilitate purification of thepolymerase. For example, His tagged polymerase can be purified from araw bacterial lysate by contacting the lysate with any suitable affinitymedium comprising bound metal ions to which the histidine residues ofthe His tag can chelate. The bound metal ions can comprise either nickelor cobalt, to which the polyhistidine-tag binds with micromolaraffinity. Suitable affinity media include Ni Sepharose (such as, forexample, that provided by GE Healthcare), NTA-agarose (such as, forexample, that provided by Qiagen), HisPur® resin (Thermo Scientific,Pierce Protein Products, Rockford, Ill.), or Talon® resin (Clontech,Mountain View, Calif.). The affinity matrix can be washed with suitablebuffers, e.g., phosphate buffers, to remove proteins that do notspecifically interact with the cobalt or nickel ion. Washing efficiencycan be improved by the addition of 20 mM imidazole. In some embodiments,the protein(s) can be eluted from the matrix. In some embodiments, theproteins can be eluted with 150-300 mM imidazole). The purity and amountof purified polymerase can be assessed using suitable methods, e.g.,SDS-PAGE, size exclusion chromatography, mass spectrometry and/orWestern blotting.

In some embodiments, the His tag can be followed by a suitable aminoacid sequence that facilitates removal of the His tag using a suitableendopeptidase. Alternatively, the His tag may be removed using asuitable exopeptidase, for example the Qiagen TAGZyme exopeptidase.

In addition to facilitating purification of the polymerase, the His tagcan also facilitate binding of the His tagged polymerase to thenanoparticle comprising one or more bound metal ions via chelationbonding. Exemplary methods of conjugation of a His tagged polymerase toa nanoparticle are described, for example, in U.S. ProvisionalApplication No. 61/184,770, filed Jun. 5, 2009.

In some embodiments, the amino acid sequence of a modified polymerasefused to a peptide sequence that encodes a stretch of amino acid acidscapable of functioning as a peptide linker to facilitate the formationof a linkage between the modified polymerase and another reactivemoiety. This peptide linker sequence can be fused to the N-terminus, theC-terminus or any suitable position between the N-terminus and theC-terminus of the modified polymerase.

In some embodiments, the peptide linker can comprise the amino acidsequence:

(SEQ ID NO: 16) LLGAAAKGAAAKGSAA.

This linker is hereinafter referred to as the “H-linker”. Without beingbound to any particular theory of operation, it has been suggested thatthis linker comprises a helix-forming peptide that can effectivelyseparate different functional domains of a fusion protein or conjugate.See, for example, Arai et al., Protein Engineering 14(8): 529-532(2001); Marqusee & Baldwin, Proc. Natl. Acad. Sci. USA, 84: 8898-8902(1987).

In some embodiments, the peptide linker can comprise the amino acidsequence: LLGGGGSGGGGSAAAGSAA (SEQ ID NO: 17). In some embodiments, thepeptide linker can comprise the amino acid sequence:MNHLVHHHHHHIE-GRHMELGTLEGS (SEQ ID NO: 51). In some embodiments, thepeptide linker can comprise the amino acid sequence: MHHHHHHKH (SEQ IDNO: 52). In some embodiments, the peptide linker can comprise the aminoacid sequence: GLNDIF-EAQKIEWHE (SEQ ID NO:53).

This linker is hereinafter referred to as the “F-linker”. See, forexample, Arai et al., Protein Engineering 14(8): 529-532 (2001); Alfthanet al., Protein Engineering 8(7): 725-731 (1995).

In some embodiments, the modified polymerase comprises a modifiedN-terminal His tagged version of a B103 polymerase, wherein the modifiedpolymerase comprises the following amino acid sequence:

(SEQ ID NO: 18)         10         20         30         40MNHLVHHHHH HIEGRHMELG TLEGSMPRKM FSCDFETTTK        50         60         70         80LDDCRVWAYG YMEIGNLDNY KIGNSLDEFM QWVMEIQADL        90        100        110        120YFHNLKFDGA FIVNWLEHHG FKWSNEGLPN TYNTIISKMG       130        140        150        160QWYMIDICFG YKGKRKLHTV IYDSLKKLPF PVKKIAKDFQ       170        180        190        200LPLLKGDIDY HAERPVGHEI TPEEYEYIKN DIEIIARALD       210        220        230        240IQFKQGLDRM TAGSDSLKGF KDILSTKKFN KVFPKLSLPM       250        260        270        280DKEIRRAYRG GFTWLNDKYK EKEIGEGMVF DVNSLYPSQM       290        300        310        320YSRPLPYGAP IVFQGKYEKD EQYPLYIQRI RFEFELKEGY       330        340        350        360IPTIQIKKNP FFKGNEYLKN SGAEPVELYL TNVDLELIQE       370        380        390        400HYEMYNVEYI DGFKFREKTG LFKEFIDKWT YVKTHEKGAK       410        420        430        440KQLAKLMLNS LYGKFASNPD VTGKVPYLKE DGSLGFRVGD       450        460        470        480EEYKDPVYTP MGVFITAWAR FTTITAAQAC YDRIIYCDTD       490        500        510        520SIHLTGTEVP EIIKDIVDPK KLGYWAHEST FKRAKYLRQK       530        540        550        560TYIQDIYAKE VDGKLIECSP DEATTTKFSV KCAGMTDTIK       570        580        590KKVTFDNFRV GFSSTGKPKP VQVNGGVVLV DSVFTIK

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 18. In some embodiments, themodified polymerase further comprises the amino acid substitution D191A,wherein the numbering is relative to SEQ ID NO: 18.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 18 and comprises any one, two, three or moreof the mutations described herein.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 18 and further comprises one or more aminoacid substitutions selected from the group consisting of: D34A, E36A,H83R, N84D, D88A, D191A, Q402A and S410G, and further comprises one ormore amino acid substitutions selected from the group consisting of:H395G, H395T, H395S, H395K, H395R, H395A, H395Q, H395W, H395Y, H395F,E396G, E396H, E396T, E396S, E396K, E396R, E396A, E396Q, E396W, E396Y,E396F, K397G, K397E, K397T, K397S, K397R, K397A, K397Q, K397W, K397Y,K397F, K405E, K405T, K4055, K405R, K405A, K405Q, K405W, K405Y, K405F,D532H, D532G, D532E, D532T, D532S, D532R, D532A, D532R, D532Q, D532W,D532Y, D532F, K534H, K534G, K534D, K534R, K534E, K534T, K534S, K534R,K534A, K534Q, K534W, K534Y and K534F, wherein the numbering is relativeto the sequence of SEQ ID NO: 18.

In some embodiments, the modified polymerase comprising the amino acidsequence of SEQ ID NO: 18 further comprises one or more amino acidsubstitutions selected from the group consisting of: D34A, E36A, H83R,N84D, D88A, D191A, Q402A and S410G, and further comprises amino acidsubstitutions at two or more positions selected from the groupconsisting of: 395, 397 and 532, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 18.

In some embodiments, the modified polymerase comprises the amino acidsequence of comprises one or more amino acid substitutions selected fromthe group consisting of: D34A, E36A, H83R, N84D, D88A, D191A, Q402A andS410G, and further comprises the amino acid substitutions K397Y andD532H, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 18.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D34A, E36A,H83R, N84D, D88A, D191A, Q402A and S410G, and further comprises theamino acid substitutions H395R and D532H, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D34A, E36A,H83R, N84D, D88A, D191A, Q402A and S410G, and further comprises two ormore amino acid substitutions selected from the group consisting of:H395R, K397Y and D532H, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 18.

In some embodiments, the modified polymerase comprises a modifiedN-terminal His tagged version of a B103 polymerase, wherein the modifiedpolymerase comprises the following amino acid sequence:

(SEQ ID NO: 19)         10         20         30         40MHHHHHHKHM PRKMFSCDFE TTTKLDDCRV WAYGYMEIGN        50         60         70         80LDNYKIGNSL DEFMQWVMEI QADLYFHNLK FDGAFIVNWL        90        100        110        120EHHGFKWSNE GLPNTYNTII SKMGQWYMID ICFGYKGKRK       130        140        150        160LHTVIYDSLK KLPFPVKKIA KDFQLPLLKG DIDYHAERPV       170        180        190        200GHEITPEEYE YIKNDIEIIA RALDIQFKQG LDRMTAGSDS       210        220        230        240LKGFKDILST KKFNKVFPKL SLPMDKEIRR AYRGGFTWLN       250        260        270        280DKYKEKEIGE GMVFDVNSLY PSQMYSRPLP YGAPIVFQGK       290        300        310        320YEKDEQYPLY IQRIRFEFEL KEGYIPTIQI KKNPFFKGNE       330        340        350        360YLKNSGAEPV ELYLTNVDLE LIQEHYEMYN VEYIDGFKFR       370        380        390        400EKTGLFKEFI DKWTYVKTHE KGAKKQLAKL MLNSLYGKFA       410        420        430        440SNPDVTGKVP YLKEDGSLGF RVGDEEYKDP VYTPMGVFIT       450        460        470        480AWARFTTITA AQACYDRIIY CDTDSIHLTG TEVPEIIKDI       490        500        510        520VDPKKLGYWA HESTFKRAKY LRQKTYIQDI YAKEVDGKLI       530        540        550        560ECSPDEATTT KFSVKCAGMT DTIKKKVTFD NFRVGFSSTG        570        580KPKPVQVNGG VVLVDSVFTI K

In some embodiments, the modified polymerase can comprise an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 19. In some embodiments, themodified polymerase further comprises the amino acid substitution D175A,wherein the numbering is relative to SEQ ID NO: 19.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 19 and comprises any one, two, three or moreof the mutations described herein.

In some embodiments, the modified polymerase comprises an amino acidsequence that is at least 85%, 90%, 95% or 99% identical to the aminoacid sequence of SEQ ID NO: 19 and further comprises any one, two, threeor more of the mutations selected from the group consisting of: D18A,E20A, H67R, N68D, D72A, D175A, Q386A, S394G, H379G, H379T, H379S, H379K,H379R, H379A, H379Q, H379W, H379Y, H379F, E379G, E380H, E380T, E380S,E380K, E380R, E380A, E380Q, E380W, E380Y, E380F, K381G, K381E, K381T,K381S, K381R, K381A, K381Q, K381W, K381Y, K381F, K389E, K389T, K389S,K389R, K389A, K389Q, K389W, K389Y, K389F, D516H, D516G, D516E, D516T,D516S, D516R, D516A, D516R, D516Q, D516W, D516Y D516F, K518H, K518G,K518D, K518R, K518E, K518T, K518S, K518R, K518A, K518Q, K518W, K518Y andK518F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 19.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D18A, E20A,H67R, N68D, D72A, D175A, Q386A and S394G, and further comprises one ormore amino acid substitutions selected from the group consisting of:H379G, H379T, H379S, H379K, H379R, H379A, H379Q, H379W, H379Y, H379F,E379G, E380H, E380T, E380S, E380K, E380R, E380A, E380Q, E380W, E380Y,E380F, K381G, K381E, K381T, K381S, K381R, K381A, K381Q, K381W, K381Y,K381F, K389E, K389T, K389S, K389R, K389A, K389Q, K389W, K389Y, K389F,D516H, D516G, D516E, D516T, D516S, D516R, D516A, D516R, D516Q, D516W,D516Y D516F, K518H, K518G, K518D, K518R, K518E, K518T, K518S, K518R,K518A, K518Q, K518W, K518Y and K518F, wherein the numbering is relativeto the sequence of SEQ ID NO: 19.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D18A, E20A,H67R, N68D, D72A, D175A, Q386A and S394G and further comprises aminoacid substitutions at two or more positions selected from the groupconsisting of: 379, 380 and 516, wherein the numbering is relative tothe sequence of SEQ ID NO: 19.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D18A, E20A,H67R, N68D, D72A, D175A, Q386A and S394G, and further comprises theamino acid substitutions K381Y and D516H, wherein the numbering isrelative to the sequence of SEQ ID NO: 19.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D18A, E20A,H67R, N68D, D72A, D175A, Q386A and S394G, and further comprises theamino acid substitutions H379R and D516H, wherein the numbering isrelative to the sequence of SEQ ID NO: 19.

In some embodiments, the modified polymerase comprises one or more aminoacid substitutions selected from the group consisting of: D18A, E20A,H67R, N68D, D72A, D175A, Q386A and S394G, and further comprises two ormore amino acid substitutions selected from the group consisting of:H379R, K381Y and D516H, wherein the numbering is relative to thesequence of SEQ ID NO: 19.

In some embodiments, one or more activities or properties of a modifiedpolymerase comprising an amino acid sequence that is at least %, 90%,95% or 99% identical to the amino acid sequence of SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 18 or SEQ ID NO: 19 can be altered (e.g., increased ordecreased) relative to the corresponding one or more activities orproperties of an exemplary reference polymerase having an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the following amino acid sequence:

(SEQ ID NO: 20) MNHLVHHHHH HIEGRHMELG TLEGSMKHMP RKMYSCAFET        70         80         90        100TTKVEDCRVW AYGYMNIEDH SEYKIGNSLD EFMAWVLKVQ       110        120        130        140ADLYFHNLKF AGAFIINWLE RNGFKWSADG LPNTYNTIIS       150        160        170        180RMGQWYMIDI CLGYKGKRKI HTVIYDSLKK LPFPVKKIAK       190        200        210        220DFKLTVLKGD IDYHKERPVG YKITPEEYAY IKNDIQIIAE       230        240        250        260ALLIQFKQGL DRMTAGSDSL KGFKDIITTK KFKKVFPTLS       270        280        290        300LGLDKEVRYA YRGGFTWLND RFKEKEIGEG MVFDVNSLYP       310        320        330        340AQMYSRLLPY GEPIVFEGKY VWDEDYPLHI QHIRCEFELK       350        360        370        380EGYIPTIQIK RSRFYKGNEY LKSSGGEIAD LWLSNVDLEL       390        400        410        420MKEHYDLYNV EYISGLKFKA TTGLFKDFID KWTYIKTTSE       430        440        450        460GAIKQLAKLM LNSLYGKFAS NPDVTGKVPY LKENGALGFR       470        480        490        500LGEEETKDPV YTPMGVFITA WARYTTITAA QACYDRIIYC       510        520        530        540DTDSIHLTGT EIPDVIKDIV DPKKLGYWAH ESTFKRAKYL       550        560        570        580RQKTYIQDIY MKEVDGKLVE GSPDDYTDIK FSVKCAGMTD        590        600KIKKEVTFEN FKVGFSRKMK PKPVQVPGGV VLVDDTFTIK

This fusion polymerase of amino acid sequence of SEQ ID NO: 20 is hereinvariously referred to as “HP1” or “HP-1”. See, e.g., U.S. ProvisionalApplication No. 61/184,770, filed Jun. 5, 2009. This referencepolymerase typically comprises a Phi-29 polymerase peptide that lacksexonuclease activity and comprises an N-terminal His-tag, an interveninglinker sequence, and the D12A and D66A mutations.

In some embodiments, the reference polymerase comprises an amino acidsequence that is at least 70%, 80%, 85%, 90%, 95%, 99% or 100% identicalto the amino acid sequence of SEQ ID NO: 20 (HP-1).

In one embodiment, the polymerase can be a fusion protein comprising theamino acid sequence of a nucleic acid-dependent polymerase (thepolymerase portion) linked to the amino acid sequence of a second enzymeor a biologically active fragment thereof (the second enzyme portion).The second enzyme portion of the fusion protein may be linked to theamino or carboxyl end of the polymerase portion, or may be insertedwithin the polymerase portion. The polymerase portion of the fusionprotein may be linked to the amino or carboxyl end of the second enzymeportion, or may be inserted within the second enzyme portion. In someembodiments, the polymerase and second enzyme portions can be linked toeach other in a manner which does not significantly interfere withpolymerase activity of the fusion or with the ability of the fusion tobind nucleotides, or does not significantly interfere with the activityof the second enzyme portion. In the fusion protein, the polymeraseportion or the second enzyme portions can be linked with at least oneenergy transfer donor moiety. The fusion protein can be a recombinantprotein having a polymerase portion and a second enzyme portion. In someembodiments, the fusion protein can include a polymerase portionchemically linked to the second enzyme portion.

In some embodiments, the polymerase can be a modified polymerase havingcertain desired characteristics, such as an evolved polymerase selectedfrom a directed or non-directed molecular evolution procedure. Theevolved polymerase can exhibit modulated characteristics or functions,such as changes in: affinity, specificity, or binding rates forsubstrates (e.g., target molecules, polymerization initiation sites, ornucleotides); binding stability to the substrates (e.g., targetmolecules, polymerization initiation sites, or nucleotides); nucleotideincorporation rate; nucleotide analog permissiveness; exonucleaseactivity (e.g., 3′→5′ or 5′→3′); rate of extension; processivity;fidelity; stability; or sensitivity and/or requirement for temperature,chemicals (e.g., DTT), salts, metals, pH, or electromagnetic energy(e.g., excitation or emitted energy). Many examples of evolvedpolymerases having altered functions or activities can be found in U.S.provisional patent application No. 61/020,995, filed Jan. 14, 2008.

Methods for creating and selecting proteins and enzymes having thedesired characteristics are known in the art, and include:oligonucleotide-directed mutagenesis in which a short sequence isreplaced with a mutagenized oligonucleotide; error-prone polymerasechain reaction in which low-fidelity polymerization conditions are usedto introduce point mutations randomly across a sequence up to about 1 kbin length (R. C. Caldwell, et al., 1992 PCR Methods and Applications2:28-33; H. Gramm, et al., 1992 Proc. Natl. Acad. Sci. USA89:3576-3580); and cassette mutagenesis in which a portion of a sequenceis replaced with a partially randomized sequence (A. R. Oliphant, etal., 1986 Gene 44:177-183; J. D. Hermes, et al., 1990 Proc. Natl. Acad.Sci. USA 87:696-700; A. Arkin and D. C. Youvan 1992 Proc. Natl. Acad.Sci. USA 89:7811-7815; E. R. Goldman and D. C. Youvan 1992Bio/Technology 10:1557-1561; Delagrave et al., 1993 Protein Engineering6: 327-331; Delagrave et al., 1993 Bio/Technology 11: 1548-155); anddomain shuffling.

Methods for creating evolved antibody and antibody-like polypeptides canbe adapted for creating evolved polymerases, and include appliedmolecular evolution formats in which an evolutionary design algorithm isapplied to achieve specific mutant characteristics. Many library formatscan be used for evolving polymerases including: phage libraries (J. K.Scott and G. P. Smith 1990 Science 249:386-390; S. E. Cwirla, et al.1990 Proc. Natl. Acad. Sci. USA 87:6378-6382; J. McCafferty, et al. 1990Nature 348:552-554) and lad (M. G. Cull, et al., 1992 Proc. Natl. Acad.Sci. USA 89:1865-1869).

Another adaptable method for evolving polymerases employs recombination(crossing-over) to create the mutagenized polypeptides, such asrecombination between two different plasmid libraries (Caren et al. 1994Bio/Technology 12: 517-520), or homologous recombination to create ahybrid gene sequence (Calogero, et al., 1992 FEMS Microbiology Lett. 97:41-44; Galizzi et al., WO91/01087). Another recombination methodutilizes host cells with defective mismatch repair enzymes (Radman etal., WO90/07576). Other methods for evolving polymerases include randomfragmentation, shuffling, and re-assembly to create mutagenizedpolypeptides (published application No. U.S. 2008/0261833, Stemmer).Adapting these mutagenesis procedures to generate evolved polymerases iswell within the skill of the art.

In some embodiments, the polymerase can be fused with, or otherwiseengineered to include, DNA-binding or other domains from other proteinsthat are capable of modulating DNA polymerase activity. For example,fusion of suitable portions of the Single-Stranded DNA Binding Protein(SSBP), thioredoxin and/or T7 DNA polymerase to bacterial or viral DNApolymerases has been shown to enhance both the processivity and fidelityof the DNA polymerase. Similarly, other groups have described efforts toengineer polymerases so as to broaden their substrate range. See, e.g.,Ghadessy et al, Nat. Biotech., 22 (6):755-759 (2004). Similarly, theconjugates of the present disclosure can optionally comprise anypolymerase engineered to provide suitable performance characteristics,including for example a polymerase fused to intact SSBP or fragmentsthereof, or to domains from other DNA-binding proteins (such as theherpes simplex virus UL42 protein.)

In some embodiments, a blend of different conjugates, each of whichcomprises a polymerase of unique sequence and characteristics, can beused according to the methods described herein. Use of such conjugateblends can additionally increase the fidelity and processivity of DNAsynthesis. For example, use of a blend of processive and non-processivepolymerases has been shown to result in increased overall read lengthduring DNA synthesis, as described in U.S. Published App. No.2004/0197800. Alternatively, conjugates comprising polymerases ofdifferent affinities for specific acceptor-labeled nucleotides can beused so as to achieve efficient incorporation of all four nucleotides.

In some embodiments, the polymerase can comprise the amino acid sequenceof any polymerase disclosed in U.S. Provisional Application No.61/242,771, filed on Sep. 15, 2009; 61/263,974, filed on Nov. 24, 2009and 61/299,919, filed on Jan. 29, 2010, or any variant thereof.

In some embodiments, the polymerases of the present disclosure can beisolated from a cell, or generated using recombinant DNA technology orchemical synthesis methods. In another embodiment, the polymerases canbe expressed in prokaryote, eukaryote, viral, or phage organisms. Inanother embodiment, the polymerases can be post-translationally modifiedproteins or fragments thereof.

In some embodiments, the polymerases of the present disclosure can berecombinant proteins that are produced by a suitable expressionvector/host cell system. The polymerases can be encoded by suitablerecombinant expression vectors carrying inserted nucleotide sequences ofthe polymerases. In some embodiments, the polymerase sequence can belinked to a suitable expression vector. The polymerase sequence can beinserted in-frame into the suitable expression vector. The suitableexpression vector can replicate in a phage host, or a prokaryotic oreukaryotic host cell. The suitable expression vector can replicateautonomously in the host cell, or can be inserted into the host cell'sgenome and be replicated as part of the host genome. The suitableexpression vector can carry a selectable marker which confers resistanceto drugs (e.g., kanamycin, ampicillin, tetracycline, chloramphenicol, orthe like), or confers a nutrient requirement. The suitable expressionvector can have one or more restriction sites for inserting the nucleicacid molecule of interest. The suitable expression vector can includeexpression control sequences for regulating transcription and/ortranslation of the encoded sequence. The expression control sequencescan include: promoters (e.g., inducible or constitutive), enhancers,transcription terminators, and secretion signals. The expression vectorcan be a plasmid, cosmid, or phage vector. The expression vector canenter a host cell which can replicate the vector, produce an RNAtranscript of the inserted sequence, and/or produce protein encoded bythe inserted sequence. The recombinant polymerase can include anaffinity tag for enrichment or purification, including a poly-amino acidtag (e.g., poly His tag), GST, and/or HA sequence tag. Methods forpreparing suitable recombinant expression vectors and expressing the RNAand/or protein encoded by the inserted sequences are well known(Sambrook et al, Molecular Cloning (1989)).

In some embodiments, the present disclosure relates to modifiedpolymerases exhibiting altered kinetics of nucleotide binding,nucleotide incorporation and/or primer extension. For example, in someembodiments, the modified polymerases of the present disclosure compriseone or more modifications resulting in a change in the kinetic behaviorof the polymerase in vitro or in vivo. For example, the modification(s)may result in a change (for example, an increase or decrease) in one ormore of the following activities or properties of the polymerase,relative to the corresponding activity or property of a referencepolymerase: specific activity as measured in a primer extension assay;specific activity as measured in a nucleotide incorporation assay(including assays for incorporation of naturally occurring nucleotidesand nucleotide analogs); exonuclease activity (including, for example,3′ to 5′ exonuclease activity); ability to bind one or more substrates(including naturally occurring nucleotides and nucleotide analogs);yield of synthesized nucleic acid product; processivity; fidelity ofnucleic acid synthesis; rate of nucleic acid synthesis; binding affinityfor one or more particular nucleotides (including naturally occurringnucleotides and nucleotide analogs); K_(m) for one or more substrates(including naturally occurring nucleotides, nucleotide analogs and/ortemplate strand); k_(cat) or V_(max), t_(po1), t⁻¹, k_(po1), or k⁻¹ forone or more nucleotides (including naturally occurring nucleotides andnucleotide analogs); residence time of one or more nucleotides(including naturally occurring nucleotides and nucleotide analogs)within one or more polymerase active sites; rate of binding for one ormore nucleotides (including naturally occurring nucleotides andnucleotide analogs); rate of nucleotide release (in either altered orunaltered state) from the polymerase active site, including, forexample, rate of product release; average template read length inpresence of nucleotides (including naturally occurring nucleotides andnucleotide analogs); nucleotide binding specificity for one or moreparticular nucleotides (including naturally occurring nucleotides andnucleotide analogs); accessibility of one or more polymerase activesites by one or more nucleotides (including naturally occurringnucleotides and nucleotide analogs); steric inhibition of nucleotideentry into a polymerase active site (including, for example, inhibitionof entry of naturally occurring nucleotides and nucleotide analogs);complementarity of a polymerase active site with one or more natural,unnatural or non-complementary nucleotides (including naturallyoccurring nucleotides and nucleotide analogs) as compared to that ofcomplementary nucleotides; ability to discriminate between correct andincorrect nucleotides (including naturally occurring nucleotides andnucleotide analogs); frequency of incorporation of non-complementarynucleotides as compared to that of complementary nucleotides (includingnaturally occurring nucleotides and nucleotide analogs); branching ratiofor one or more nucleotides (including naturally occurring nucleotidesand nucleotide analogs); stability, for example at elevatedtemperatures; observed performance in a particular biological assay;complementarity with one or more natural or non-natural features of anucleotide (including naturally occurring nucleotides and nucleotideanalogs); tolerance of the polymerase for various chemical and/orphysical stresses, as well as the stability of the polymerase under agiven set of conditions, including photostability and chemicalstability; tolerance for the presence of labels (including both organiclabels, e.g., dyes, and inorganic labels, e.g., nanoparticles); andphotostability.

In some embodiments, the modified polymerases and/or polynucleotides ofthe present disclosure can exhibit an altered kinetic behavior with oneor more nucleotide substrates, including, for example, labelednucleotide analogs, relative to an unmodified counterpart. For example,in some embodiments, the modified polymerase can exhibit alteredkinetics of polymerization, including, for example, polymerization ofsubstrates comprising labeled nucleotide analogs. In some embodiments,the modified polymerase can exhibit an altered K_(m) value for asubstrate, particularly a labeled nucleotide analog. In someembodiments, the polymerase can be engineered to exhibit alteredK_(cat)/K_(m) and/or V_(max)/K_(m) for a substrate, particularly alabeled nucleotide analog. In some embodiments, the K_(cat)/K_(m), theV_(max)/K_(m), or both, are increased relative to the wild typepolymerase. In some embodiments, the modified polymerase can exhibit analtered K_(m), K_(D), t⁻¹, t_(po1), nucleotide residence time for one ormore nucleotides, for example a labeled nucleotide analog.

The kinetic activity of an enzyme can be modeled using various methods.Some exemplary theories of enzyme kinetics and associated models can befound, for example, in Berg at al., Biochemistry, 5th Ed. (W.H. Freeman,2007); Fersht, Enzyme Structure & Mechanism, 5th Ed. (W.H. Freeman,1985). Without being bound by any particular theory, theMichaelis-Menten theory provides an non-limiting exemplary theoreticalmodel for enzymatic activity. This model is based on the assumption thatan enzyme-substrate reaction typically proceeds as follows:

Where E=free enzyme; S=free substrate; P=product and ES=enzyme-substratecomplex, k₁ is the rate constant for the association of substrate (S)and enzyme (E) to form an enzyme-substrate complex (ES); k₂ is the rateconstant for the dissociation of the enzyme-substrate complex (ES) intoproduct (i.e., altered substrate) and free enzyme, and k⁻¹ is the rateconstant for the dissociation of the enzyme-substrate complex, ES, intounaltered substrate and enzyme.

The equilibrium dissociation constant for the dissociation of theenzyme-substrate complex, ES, into unaltered substrate and enzyme,K_(D), can be determined as the ratio of k⁻¹ and k₁. Mathematically,this relationship can be represented as:K_(D) =k ⁻¹ /k ₁

K_(D) can also be expressed as a ratio of the equilibrium concentrationsof enzyme, substrate and enzyme-substrate complex:K_(D)=[E][S]/[ES]

The Michaelis-Menten equation describes the rate of initial enzymaticactivity, ν, as a function of substrate concentration:ν=V_(max)[S]/(K_(m)+[S])

where the variables can be defined as follows:

ν is the reaction rate, measured as amount of product formed per timeinterval.

[S] is the concentration of uncombined, i.e., free, substrate.

V_(max) is maximal enzyme velocity, extrapolated to maximum substrateconcentrations, i.e., rate when enzyme is saturated with substrate (alsoreferred to as enzymatic “rate”); and

K_(m) is the so-called Michaelis-Menten constant, can be defined as(k₂+k⁻¹)/k₁. K_(m) is typically related to the dissociation constant,and provides an indication of the affinity of the enzyme for aparticular substrate (and hence the stability of the enzyme-substratecomplex). In the most simple case, when k₂<<k₁, product formation isvery slow compared to the rate of formation of enzyme-substrate complex(ES), i.e., product formation is the rate limiting step, then K_(m) canequal or approximate the equilibrium dissociation constant of theenzyme-substrate complex, K_(D), and thus can describe the bindingaffinity of the substrate for the enzyme. Typically, K_(m) can bemeasured as the substrate concentration at which the reaction rate ishalf the maximum enzyme velocity, V_(max).

V_(max), k_(cat) and E_(T), the total enzyme concentration (i.e., theconcentration of active sites), can be mathematically related in thefollowing equation: k_(cat)=V_(max)/E_(T)

For a polymerase reaction involving incorporation of a nucleotide ontothe end of an extending nucleic acid molecule, the enzyme-substrateinteractions can be more complex because they involve enzymaticinteractions with the nucleic acid molecule as well as the nucleotide. Atypical exemplary reaction can be represented by the following pathway:

A more detailed representation of this exemplary theoretical model isalso depicted in FIG. 1. As depicted above and in FIG. 1, the nucleotideincorporation reaction pathway can typically begin with the initialassociation of the enzyme with a nucleic acid molecule comprising “n”nucleotide subunits (represented above and in FIG. 1 as “NA.”) to forman enzyme-nucleic acid complex, represented as E•NA_(n). The E•NA_(n)complex can also associate with a nucleotide, represented above and inFIG. 1 as “N—P” although this representation is in no way intended torestrict the nucleotide to any particular structure or any particularnumber of phosphate groups, as opposed to other forms of nucleotides andnucleotide analogs, which are also included. This association of theE•NA_(n) complex with the nucleotide results in the formation of aternary complex, represented as E•NA_(n)•N—P. This ternary complex canin some instances dissociate with rate constant k⁻¹ to yield anenzyme-nucleic acid complex, E•NA_(n), comprising enzyme bound tonucleic acid, and unaltered nucleotide, N—P (a so-called “non-productiveincorporation”). Alternatively, in some instances the ternary complexcan undergo a productive incorporation wherein the nucleic acid moleculeis extended by one nucleotide moiety with the liberation of apolyphosphate moiety. During this productive incorporation, thepolymerase can undergo a conformational change from the so-called “open”form to the “closed” form wherein the nucleotide is positioned in thepolymerase active site; the resulting complex comprising the polymerasein “closed” conformation is represented as E*•NA_(n)•N—P. This complexcan give rise to an intermediate complex comprising the “closed”-formpolymerase, an extended nucleic acid and a polyphosphate group; thisintermediate complex can be represented as E*•NA_(n+1)•(Pi)_(n), whereinthe polyphosphate moiety is represented herein and in FIG. 1 as(Pi)_(n). The polymerase of the complex can undergo a reverseconformational change from the “closed” to “open” form, resulting in acomplex comprising “open”-form polymerase, extended nucleic acid andpolyphosphate moiety (E•NA_(n+1)•(Pi)_(n)). Release of the polyphosphatemoiety ((Pi)_(n)) results in a complex comprising polymerase andextended nucleic acid, which can in some instances associate withanother nucleotide to form a ternary complex that can undergo anotherproductive incorporation.

Although the polyphosphate moiety is represented herein and in FIG. 1 as(Pi), this representation is in no way intended to restrict thepolyphosphate moiety to monophosphates or inorganic phosphate. In someembodiments, for example, the nucleotide can be a phosphate-labelednucleotide analog and the liberated moiety can comprise any one, two,three, four, five, six, seven, eight, nine, ten or more phosphate groupslinked to a label, e.g., a dye moiety. The number of phosphate groups inthe liberated polyphosphate will depend on the number of phosphates inthe polyphosphate chain of the nucleotide substrate. A more completedescription of this theoretical model of polymerase activity can befound, for example, in Patel et al., “Pre-Steady-State Kinetic Analysisof Processive DNA Replication Including Complete Characterization of anExonuclease-Deficient Mutant” Biochemistry 30:511-525 (1991).

In FIG. 1, the step involving productive incorporation is furtherdepicted as separated into three sub-steps, with the first sub-stepinvolving formation of the ternary complex, E•NA_(n)•N—P, and the secondsub-step involves the conversion of E•NA_(n)•N—P to E*•NA_(n)•N—P_(n),wherein the enzyme has undergone a conformational change from “open”form (E) to “closed” form (E*). The third sub-step, the so-called“chemistry” step, typically involves the conversion of E*•NA_(n)•N—P toE*•NA_(n+1)•(Pi)_(n), wherein the nucleic acid is extended by a singlenucleotide moiety, with the formation of a polyphosphate by-product,denoted as (Pi)_(n). In some embodiments, the E′•NA_(n+1)•(Pi)_(n)complex can dissociate to form free enzyme, extended nucleic acid, andfree polyphosphate (E+NA_(n+1)+(Pi)_(n); alternatively, thepolyphosphate may dissociate from the complex while the enzyme remainsassociated with the extended nucleic acid and can in some embodimentscatalyze a subsequent nucleotide incorporation. FIG. 1 also depicts awindow wherein FRET activity can occur using embodiments wherein anysuitable combination of components selected from the group consisting ofthe nucleic acid molecule, the nucleotide, the primer and the polymeraseare labeled with moieties capable of undergoing FRET with each other. Insome embodiments, the polymerase is labeled with one member of a FRETdonor:acceptor pair and the incoming nucleotide is labeled with anothermember of the FRET pair.

In some embodiments, the above reaction involves a polymerase, anysuitable nucleic acid molecule (represented above as NA_(n)) and anysuitable nucleotide. In some embodiments, the nucleotide can be apolyphosphate-comprising nucleotide, represented above as N—P. Anysuitable polyphosphate-comprising nucleotide can be used, including, forexample labeled deoxynucleotide polyphosphates comprising tri-, tetra-,penta-hexa-phosphate, hepta-phosphate, octa-phosphate, nona-phosphate,deca-phosphate, and undeca-phosphate moieties, as well asdeoxynucleotide polyphosphates comprising twelve or more phosphategroups.

Theoretically, each step of this reaction pathway is associated withspecific rate constants, dissociation constants, and associated kineticparameters. In one exemplary embodiment, one or more of these kineticparameters can be measured in a “stopped-flow” assay using suitabletechniques. See, for example, M. P. Roettger, Biochemistry 47:9718-9727(2008); M. Bakhtina Biochemistry 48:3197-320 (2009); Ahn et al,Biochemistry 36:1100-1107 (1997).

Without intending to be bound to any particular theory of reactionmechanism, it can be surmised that in some cases k⁻¹, which indicatesthe rate of dissociation of the ternary complex (E•NA_(n)•N—P) intoenzyme-nucleic acid complex (E•NA_(n)) and free, unaltered nucleotide(N—P), is significantly lower than k_(po1), which indicates the rate atwhich the ternary complex (E•NA_(n)•N—P) converts to the intermediatecomplex (E*•NA_(n+1)•(Pi)_(n)), then it can be expected thatnon-productive incorporation events will predominate over productiveincorporation events. Conversely, when k_(po1) is significantly lowerthan k⁻¹ then it can be expected that productive incorporations willpredominate over non-productive incorporations.

In some embodiments, the polymerases, nucleotides, and reactionconditions, can be screened for their suitability for use in thedisclosed nucleotide binding, nucleotide incorporation and/or primerextension methods, using well known screening techniques. For example,the suitable polymerase may be capable of binding nucleotides and/orincorporating nucleotides. In some embodiments, the reaction kineticsfor nucleotide binding, association, incorporation, and/or dissociationrates, can be determined using rapid kinetics techniques (e.g.,stopped-flow or quench flow techniques). Using stopped-flow or quenchflow techniques, the binding kinetics of a nucleotide can be estimatedby calculating the 1/k_(d) value. Stopped-flow techniques which analyzeabsorption and/or fluorescence spectroscopy properties of the nucleotidebinding, incorporation, or dissociation rates to a polymerase are wellknown in the art (Kumar and Patel 1997 Biochemistry 36:13954-13962; Tsaiand Johnson 2006 Biochemistry 45:9675-9687; Hanzel, U.S. publishedpatent application No. 2007/0196846). Other methods include quench flow(Johnson 1986 Methods Enzymology 134:677-705), time-gated fluorescencedecay time measurements (Korlach, U.S. Pat. No. 7,485,424), plate-basedassays (Clark, U.S. published patent application No. 2009/0176233), andX-ray crystal structure analysis (Berman 2007 EMBO Journal 26:3494).Nucleotide incorporation by a polymerase can also be analyzed by gelseparation of the primer extension products.

In some embodiments, the modified polymerase exhibits altered (e.g.,increased or decreased) levels of primer extension activity. Forexample, the modified polymerase may exhibit increased primer extensionactivity in the presence of labeled nucleotides, relative to anunmodified counterpart. In some embodiments, the primer extensionactivity of the modified polymerase is at least about 5%, 10%, 25%,37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%,5,000% or 10,000% of the primer extension activity of a referencepolymerase under identical reaction conditions. In some embodiments, thereference polymerase is the unmodified counterpart of the modifiedpolymerase. In some embodiments, the reference polymerase is a Phi-29polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the reference polymerase is a B103 polymerase having theamino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant form of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the nucleotide substrate is a natural nucleotide. In someembodiments, the nucleotide substrate is a labeled nucleotide analog.The polymerase can be at least 80%, 85%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In someembodiments, the modified polymerase comprises one or more amino acidsubstitutions at positions selected from the group consisting of: 2, 9,12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375,376, 377, 380, 383, 384, 385, 455, 507 and 509, or any combinationsthereof. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

The primer extension activity can be measured using any suitable assaythat provides a quantitative indication of the amount of extensionproduct obtained using defined reaction conditions comprising a knownconcentration of polymerase. Regardless of which assay is used,differences in primer extension activity between two samples, whenobtained using identical reaction conditions, can be evaluated by simplycomparing levels of observed primer activity obtained from each sample.Optionally, the observed primer extension activity can normalized foramount of polymerase by dividing the amount of incorporatedradioactivity by the polymerase concentration in the reaction mixture,to allow comparison between reactions containing different polymeraseconcentrations.

In one exemplary embodiment, the primer extension activity of apolymerase can be measured using a radiometric assay that measuresincorporation of a radioactively labeled nucleotide into acid-insolublematerial in a polymerase reaction. The amount of incorporatedradioactivity indicates the total number of nucleotides incorporated.See, e.g., Wu et al., Gene Biotechnology, 2nd Ed., CRC Press; Sambrook,J., Fritsch, E F, and Maniatis, T. (1989) Molecular Cloning A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

In another exemplary embodiment, levels of primer extension activity ina sample can be measured by monitoring the fluorescence intensity changeover time during extension of a fluorescein-labeled hairpinoligonucleotide. One such exemplary assay is described in Example 13,herein.

In another exemplary embodiment, the primer extension activity can bequantified by quantifying the amount of pyrophosphate liberated afterperforming primer extension under standard tolerance assay conditionsfor 5 minutes. One such exemplary assay is described in Example 5.

In another exemplary embodiment, the primer extension activity can bequantified by measuring the fraction of extended primer within apopulation of primer-template duplexes. One such exemplary assay usingstandard photostability assay conditions is provided in Example 6. Inthis exemplary embodiment, the template comprised a radioactive (³²P)moiety or fluorescent (TAMRA) label to permit visualization ofpolymerase reaction products (e.g., extended primer). Primer extensionproducts were resolved on a gel, and the primer extension activity wasquantified as the proportion (%) of extended primer relative to totalstarting primer by adding the intensities of all bands observed within asingle lane as measured by densitometric analysis.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) K_(m) value for any particular nucleotidesubstrate of interest, for example a labeled nucleotide analog, relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a K_(m) value for a particular nucleotide substratethat is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%,150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as theK_(m) value of a reference polymerase for the same nucleotide substrate.In some embodiments, the reference polymerase is the unmodifiedcounterpart of the modified polymerase. In some embodiments, thereference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant form of the polymerase having the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. The polymerase can be at least 80%, 85%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO:8. In some embodiments, the modified polymerase comprises one or moreamino acid substitutions at positions selected from the group consistingof: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373,374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, or anycombinations thereof. In some embodiments, the modified polymerasecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7 and further includes amino acid mutations at any one, two,three or more positions selected from the group consisting of: 2, 9, 12,14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187,195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339,357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511,526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. In some embodiments, the modified polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase comprises one or moremodifications that alter the t₁ value of the modified polymerase for agiven nucleotide, for example a labeled nucleotide analog, as comparedto the t⁻¹ value of an unmodified counterpart for the same nucleotide.In some embodiments, the t⁻¹ value can be defined as the average timerequired for the nucleotide (e.g., a labeled nucleotide analog) todissociate in unaltered form from the enzyme-template-nucleotide ternarycomplex (E•NA_(n)•N—P) during nucleic acid polymerization, wherein thedissociation can be represented through the following reaction:

Typically, t⁻¹ can be calculated as the reciprocal of k⁻¹, the rateconstant for the dissociation reaction, and thus t⁻¹ can bemathematically represented as 1/k⁻¹. In some embodiments, the modifiedpolymerase comprises one, two or more modifications that increase thet⁻¹ value of the polymerase for a particular nucleotide substrate,particularly a labeled nucleotide analog. Such increase in the t⁻¹ valuetypically correlates with a decreased rate of dissociation of theenzyme-template-nucleotide ternary complex, which can in some casesincrease the duration during which the nucleotide remains associatedwith the polymerase and template in the ternary complex. This can behelpful in various biological applications, including nucleic acidsequencing, which require or otherwise involve visualization of thelabeled nucleotide analog when in proximity with or bound to thepolymerase active site.

A variety of methods are available to measure the rate of nucleotidedissociation, k⁻¹ (and the corresponding t⁻¹ value). Typically, the k⁻¹value is measured using stopped-flow fluorescence measurements, andfitting the resulting fluorescence traces to a single exponentialfunction of the form:Fluorescence=A ₁ *e ^(−k) ⁻¹ ^(*t)+C   [Equation (1)]

where A₁ represents the corresponding fluorescence amplitude, C is anoffset constant, and k⁻¹ is the observed rate constant for thefluorescence transition. See, e.g., Bakhtina, Biochemistry 48:3197-3208(2009).

In one exemplary method, a non-extendible primed template is formed byannealing a primer comprising a dideoxynucleotide at its 3′ end with atemplate oligonucleotide that further protrudes a few nucleotidesdownstream from the 3′ end of the primer, and comprises a donorfluorophore at its 5′ end. This non-extendible primed template is thencontacted with a nucleotide comprising an acceptor fluorophore attachedto the terminal phosphate group (also referred to herein as the “omega”phosphate group) to form a ternary complex. The ternary complex is thencontacted with unlabeled (“cold”) nucleotides, and the dissociation ofthe omega-labeled nucleotide from the ternary complex is monitored as afunction of donor fluorescence where typically dissociation iscorrelated with an increase in donor fluorescence. The fluorescencetraces are then fitted into a function of the form of Equation (1) todetermine the k⁻¹ and the corresponding t⁻¹ values for the polymerase.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) t⁻¹ value for a given nucleotide substrate, forexample a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits at_(po1) value for a particular nucleotide substrate that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000% as high as the t⁻¹ value of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or anyother variant form of the polymerase having the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the modified polymerase is at least80%, 85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and509, or any combinations thereof.

In one exemplary assay, the t⁻¹ value of the modified polymerase ismeasured with respect to an exemplary labeled nucleotide analogcomprising a fluorophore, e.g., an Alexa Fluor 647 moiety, attached tothe terminal phosphate of a deoxynucleotide tetraphosphate via asuitable linker. Some non-limiting examples of labeled nucleotides andmethods of using such nucleotides in polymerase-based applications canbe found, inter alia, in U.S. Pat. No. 7,244,566 issued Jul. 17, 2007;U.S. Pat. No. 7,223,541 issued May 29, 2007; U.S. Pat. No. 7,052,839issued May 30, 2006; U.S. Pat. No. 7,244,566 issued Jul. 17, 2007; U.S.Pat. No. 7,393,640 issued Jul. 1, 2008; U.S. Pat. No. 7,033,762 issuedApr. 25, 2006; U.S. Pat. No. 7,256,019 issued Aug. 14, 2007; U.S. Pat.No. 7,041,812 issued May 9, 2006; U.S. Pat. No. 7,452,698 issued Nov.18, 2008 and U.S. Pat. No. 7,078,499 issued Jul. 18, 2006; as well as inU.S. Published Application Nos. 2004/0048300 published Mar. 11, 2004;2008/0132692 published Jun. 5, 2008; 2009/0081686, published Mar. 26,2009; 2008/0131952, published Jun. 5, 2008; and 2007/0292679, publishedDec. 20, 2007.

In some embodiments, the modified polymerase has a t⁻¹ value for anucleotide substrate that is greater than or equal to the t⁻¹ value forthe same nucleotide substrate of a Phi-29 polymerase comprising theamino acid sequence of SEQ ID NO: 1 and further comprising an amino acidsubstitution at one or more residues selected from the group consistingof: 132, 250, 342, 373, 375, 379, 380, 383, 510 and 512, wherein thenumbering is relative to the amino acid of SEQ ID NO: 1. In someembodiments, the modified polymerase has a t⁻¹ value for a givennucleotide that is greater than or equal to the t⁻¹ for the samenucleotide of a Phi-29 polymerase comprising the amino acid sequence ofSEQ ID NO: 1 and further comprising one or more amino acid substitutionsselected from the group consisting of: K132A, V250A, L342G, T373R,E375Y, T373R, K379A, Q380A, D510H and K512Y.

In some embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase is at least 80%, 85%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 7 and can in someembodiments comprise an amino acid substitutions at one or morepositions selected from the group consisting of: 2, 9, 12, 58, 59, 63,129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380,383, 384, 385, 455, 507 and 509, or any combinations thereof. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

Another useful parameter of enzymatic activity is k_(cat), also known asthe turnover number. Typically, k_(cat) equals the number of times eachenzyme site converts substrate to product per unit time, i.e., themaximal number of molecules of substrate converted to product per enzymeactive site per unit time (e.g., second).

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) k_(cat) value for a given nucleotide substrate,for example a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits ak_(cat) value for a particular nucleotide substrate that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000% as high as the k_(cat) value of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant form of the polymerase having the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase comprises one or more amino acid substitutions at positionsselected from the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166,246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384,385, 455, 507 and 509, or any combinations thereof. In some embodiments,the modified polymerase comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 7 and further includes amino acidmutations at any one, two, three or more positions selected from thegroup consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129,147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252,256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387,389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493,494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552,555, 567, 569 and 572, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the modificationscan include deletions, additions and substitutions. The substitutionscan be conservative or non-conservative substitutions. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 7 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) specificity for a given nucleotide substrate,for example a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits aspecificity for a particular nucleotide substrate that is at least about5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%,750%, 1,000%, 5,000% or 10,000% as high as the specificity of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant form of the polymerase having the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase comprises one or more amino acid substitutions at positionsselected from the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166,246, 247, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385,455, 507 and 509, or any combinations thereof.

Without being bound to any particular theory, according to some theoriesthe specificity of the enzyme can be defined mathematically as the ratiok_(cat)/K_(m)=k_(cat)·k₁/(k⁻¹+k₂). It has the dimension M⁻¹s⁻¹, withlarge values typically indicating high specificity. In some embodiments,the modified polymerase exhibits an altered (e.g., increased ordecreased) k_(cat)/K_(m) ratio for a given nucleotide substrate, forexample a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits ak_(cat)/K_(m) ratio for a particular nucleotide substrate that is atleast about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%,250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as the k_(cat)/K_(m)ratio of a reference polymerase for the same nucleotide substrate. Insome embodiments, the reference polymerase is the unmodified counterpartof the modified polymerase. In some embodiments, the referencepolymerase is a Phi-29 polymerase having the amino acid sequence of SEQID NO: 1. In some embodiments, the reference polymerase is a B103polymerase having the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8. In some embodiments, the nucleotide substrate is anatural nucleotide. In some embodiments, the nucleotide substrate is alabeled nucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and509, or any combinations thereof. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 70%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes amino acid mutations atany one, two, three or more positions selected from the group consistingof: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176,185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302,310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392,399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503,507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and572, wherein the numbering is relative to the amino acid sequence of SEQID NO: 7. In some embodiments, the modifications can include deletions,additions and substitutions. The substitutions can be conservative ornon-conservative substitutions. In some embodiments, the modifiedpolymerase comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 7 and further includes any one, two, three or more amino acidmutations selected from the group consisting of: T365G, T365F, T365G,T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H, H370G, H370T,H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F, E371G, E371H,E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F, K380E,K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F, A481E, A481F,A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W, A481Y, D507H,D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q, D507W, D507Y,D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S, K509R, K509A,K509Q, K509W, K509Y and K509F, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7. Optionally, the modified polymerasecan further include one or more mutations reducing 3′ to 5′ exonucleaseactivity selected from the group consisting of: D9A, E11A, E11I, T12I,H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 7.Optionally, this modified polymerase comprises the amino acidsubstitution H370R.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) V_(max)/K_(m) value for a given nucleotidesubstrate, for example a labeled nucleotide analog, relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits a V_(max)/K_(m) value for a particular nucleotide substratethat is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%,150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as theV_(max)/K_(m) value of a reference polymerase for the same nucleotidesubstrate. In some embodiments, the reference polymerase is theunmodified counterpart of the modified polymerase. In some embodiments,the reference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotide substrateis a natural nucleotide. In some embodiments, the nucleotide substrateis a labeled nucleotide analog. In some embodiments, the modifiedpolymerase comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or any other variant of the polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase comprises one or more amino acid substitutions at positionsselected from the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166,246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384,385, 455, 507 and 509, or any combinations thereof. In some embodiments,the modified polymerase comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 7 and further includes amino acidmutations at any one, two, three or more positions selected from thegroup consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129,147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252,256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387,389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493,494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552,555, 567, 569 and 572, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the modificationscan include deletions, additions and substitutions. The substitutionscan be conservative or non-conservative substitutions. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 7 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase comprises one or moremodifications that alter the t_(po1) value of the modified polymerasefor a given nucleotide, for example a labeled nucleotide analog, ascompared to the t_(po1) value of an unmodified counterpart for the samenucleotide. The t_(po1) value can be defined as the average time for theincorporation reaction to occur from the enzyme-template-nucleotideternary complex according to the following reaction:

Typically, t_(po1) can be calculated as the reciprocal of k_(po1), therate constant for the above reaction, and thus t_(po1) can bemathematically represented as 1/k_(po1). In some embodiments, themodified polymerase comprises one, two or more modifications thatdecrease the t_(po1) value of the polymerase for a particular nucleotidesubstrate, particularly a labeled nucleotide analog. Such decrease inthe t_(po1) value typically correlates with an increased rate of theforward reaction in which the enzyme-template-nucleotide ternary complexundergoes a productive incorporation, thereby producing an extendednucleic acid molecule. This increase in forward rate can be helpful invarious biological applications, including nucleic acid sequencing,which require or otherwise involve visualization of the labelednucleotide analog when in proximity with or bound to the polymeraseactive site.

A variety of methods are available to measure k_(po1) (and thecorresponding t_(po1) value). In one exemplary method as taught in MPRoettger (2008 Biochemistry 47:9718-9727); M. Bakhtina 2009 Biochemistry48:3197-320), the k_(po1) value of a polymerase can be measured in astopped-flow experiment by fitting the fluorescence trace data to ageneral double exponential function of the form:Fluorescence=A ₁ *e ^(−k) ₁ *t+A ₂ *e ^(−k) ^(po1) ^(*t)+C  [Equation(2)]

where A₁ and A2 represent corresponding fluorescence amplitudes, C is anoffset constant, and k₁ and k_(po1) are the observed rate constants forthe fast and slow phases of the reaction, respectively. Plotting theconcentration dependence of the rates and amplitudes for the fast andslow phases can afford a definition of the rate constants k₁ and k_(po1)in a two-step binding process.

In one exemplary stopped-flow method, the an extendible primed templateis formed by annealing a primer with a template oligonucleotide thatextends a further few nucleotides downstream from the 3′ end of theprimer, and comprises a donor fluorophore at its 5′ end. This extendibleprimed template is contacted with a nucleotide comprising an acceptorfluorophore attached to the terminal (“omega”) phosphate group toinitiate the nucleotide incorporation reaction. The reaction progress ismonitored as a function of donor fluorescence where typically thereaction progress is correlated with an initial dip in donorfluorescence followed by a recovery in donor fluorescence. Based onthese fluorescence measurements, the k_(po1) value can be determined byfitting the fluorescence traces to a function having the form ofEquation (2), and the t_(po1) value can be calculated as the reciprocalof the k_(po1) value.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) t_(po1) value for a given nucleotide substrate,for example a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits at_(po1) value for a particular nucleotide substrate that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000% as high as the t_(po1) value of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7 and further includes amino acid mutationsat any one, two, three or more positions selected from the groupconsisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147,166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256,300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389,390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494,497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555,567, 569 and 572, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7. In some embodiments, the modifications caninclude deletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes any one, two, three ormore amino acid mutations selected from the group consisting of: T365G,T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H,H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F,E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y,E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W,A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q,D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S,K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. Optionally, themodified polymerase can further include one or more mutations reducing3′ to 5′ exonuclease activity selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, this modified polymerase comprises the aminoacid substitution H370R.

Any suitable method of measuring t_(po1) may be used. In one exemplaryassay, the t_(po1) value of the modified polymerase is measured withrespect to an exemplary labeled nucleotide analog comprising an AlexaFluor 647 moiety attached to the terminal (“omega”) phosphate of adeoxynucleotide tetraphosphate via a 6-carbon linker. See, for example,U.S. Pat. No. 7,041,812, issued May 9, 2006.

In some embodiments, the modified polymerase has a t_(po1) value for anucleotide that is less than or equal to the t_(po1) value for the samenucleotide of a Phi-29 polymerase comprising the amino acid sequence ofSEQ ID NO: 1 and further comprising an amino acid substitution at one ormore positions selected from the group consisting of: 132, 250, 342, 373375, 379, 380, 383, 510 and 512, wherein the numbering is relative tothe amino acid of SEQ ID NO: 1. In some embodiments, the modifiedpolymerase has a t_(po1) value for a given nucleotide that is greaterthan or equal to the t_(po1) value for the same nucleotide of a Phi-29polymerase comprising the amino acid sequence of SEQ ID NO: 1 andfurther comprising one or more amino acid substitutions selected fromthe group consisting of: K132A, V250A, L342G, T373R, E375Y, K379A,Q380A, D510H and K512Y.

In some embodiments, the ratio of t⁻¹/t_(po1) values for the modifiedpolymerase is at least about 0.5, more typically at least about 1.0,even more typically at least about 1.2, 1.3, 1.5, 1.75, 2.0, 2.5, 5.0,7.5, 10, 12.5, 25, 50, or 100. In some embodiments, the modifiedpolymerase exhibits an altered (e.g., increased or decreased) ratio oft⁻¹/t_(po1) values for a given nucleotide substrate, for example alabeled nucleotide analog, relative to an unmodified counterpart. Insome embodiments, the modified polymerase exhibits a t⁻¹/t_(po1) ratiofor a particular nucleotide substrate that is at least about 5%, 10%,25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%,1,000%, 5,000% or 10,000% as high as the t⁻¹/t_(po1) ratio of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 80%, 85%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In someembodiments, the modified polymerase comprises one or more amino acidsubstitutions at positions selected from the group consisting of: 2, 9,12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375,376, 377, 380, 383, 384, 385, 455, 507 and 509, or any combinationsthereof. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In one embodiment, stopped-flow techniques can be used to screen andselect mutant polymerases having a t_(po1) value (e.g., 1/k_(po1)) for aparticular labeled nucleotide that is less than the t⁻¹ (e.g., 1/k⁻¹)value of the polymerase for the same labeled nucleotide. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, themodified polymerase comprises one or more amino acid substitutions atpositions selected from the group consisting of: 2, 9, 12, 58, 59, 63,129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380,383, 384, 385, 455, 507 and 509, or any combinations thereof. In someembodiments, the polymerase comprises the amino acid sequence of SEQ IDNO: 8, and optionally further comprises the amino acid substitutionH370R. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

For example, some Phi-29 or B103 polymerases (wild type or mutant)exhibit t_(po1) values which are less than t⁻¹ values, in the presenceof nucleotide tetraphosphate or hexaphosphate molecules.

In another embodiment, polymerases can be modified by binding it to achemical compound or an antibody, in order to inhibit nucleotideincorporation.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) ratio of k_(cat)/K_(d) value values for a givennucleotide substrate, for example a labeled nucleotide analog, relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a k_(cat)/K_(d) ratio for a particular nucleotidesubstrate that is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%,110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% ashigh as the k_(cat)/K_(d) ratio of a reference polymerase for the samenucleotide substrate. In some embodiments, the reference polymerase isthe unmodified counterpart of the modified polymerase. In someembodiments, the reference polymerase is a Phi-29 polymerase having theamino acid sequence of SEQ ID NO: 1. In some embodiments, the referencepolymerase is a B103 polymerase having the amino acid sequence of SEQ IDNO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotidesubstrate is a natural nucleotide. In some embodiments, the nucleotidesubstrate is a labeled nucleotide analog.

In some embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, themodified polymerase comprises one or more amino acid substitutions atpositions selected from the group consisting of: 2, 9, 12, 58, 59, 63,129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380,383, 384, 385, 455, 507 and 509, or any combinations thereof. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the polymerase can be selected to exhibit a reducedK_(sub) for a substrate, particularly a labeled nucleotide analog. Insome embodiments, the polymerase can comprise one or more mutationsresulting in altered K_(cat)/K_(sub) and/or V_(max)/K_(sub) for aparticular labeled nucleotide. In some embodiments, the K_(cat)/K_(sub),the V_(max)/K_(sub), or both, are increased as compared to the wild typepolymerase.

In some embodiments, the modified polymerase exhibits a K_(cat)/K_(sub)and/or V_(max)/K_(sub) ratio for a particular nucleotide substrate thatis at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%,200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as theK_(cat)/K_(sub) and/or V_(max)/K_(sub) ratio of a reference polymerasefor the same nucleotide substrate. In some embodiments, the referencepolymerase is the unmodified counterpart of the modified polymerase. Insome embodiments, the reference polymerase is a Phi-29 polymerase havingthe amino acid sequence of SEQ ID NO: 1. In some embodiments, thereference polymerase is a B103 polymerase having the amino acid sequenceof SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, thenucleotide substrate is a natural nucleotide. In some embodiments, thenucleotide substrate is a labeled nucleotide analog.

In some embodiments, the modified polymerase comprises the amino acidsequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, themodified polymerase comprises one or more amino acid substitutions atpositions selected from the group consisting of: 2, 9, 12, 58, 59, 63,129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380,383, 384, 385, 455, 507 and 509, or any combinations thereof. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to exhibiteither increased or decreased residence times for one or morenucleotides, particularly for a labeled nucleotide analog. The residencetime indicates the duration for which the nucleotide dwells within theactive site of the polymerase.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) residence for a given nucleotide substrate, forexample a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits aresidence time for a particular nucleotide substrate that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000% as high as the residence time of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7 and further includes amino acid mutationsat any one, two, three or more positions selected from the groupconsisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147,166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256,300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389,390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494,497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555,567, 569 and 572, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7. In some embodiments, the modifications caninclude deletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes any one, two, three ormore amino acid mutations selected from the group consisting of: T365G,T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H,H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F,E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y,E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W,A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q,D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S,K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. Optionally, themodified polymerase can further include one or more mutations reducing3′ to 5′ exonuclease activity selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, this modified polymerase comprises the aminoacid substitution H370R.

In some embodiments, the modified polymerase has a residence time for aparticular nucleotide substrate that is between about 20 msec and about300 msec, typically between about 55 msec and about 100 msec. In someembodiments, the residence time of the selected, mutated, modified,evolved or otherwise engineered polymerase for the nucleotide substratecan be between about 1.5 and about 4 times the residence time of areference polymerase for the same nucleotide substrate. Some exemplarypolymerases exhibiting altered residence times for labeled nucleotidesare disclosed in U.S. Pub. No. 20080108082, published May 8, 2008.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) 1/(k_(po1)+k⁻¹) value for a given nucleotidesubstrate, for example a labeled nucleotide analog, relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits a 1/(k_(po1)+k⁻¹) value for a particular nucleotide substratethat is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%,150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as the1/(k_(po1)+k⁻¹) value of a reference polymerase for the same nucleotidesubstrate. In some embodiments, the reference polymerase is theunmodified counterpart of the modified polymerase. In some embodiments,the reference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotide substrateis a natural nucleotide. In some embodiments, the nucleotide substrateis a labeled nucleotide analog. In some embodiments, the modifiedpolymerase comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or any other variant of the polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase comprises one or more amino acid substitutions at positionsselected from the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166,246, 247, 339, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383,384, 385, 455, 507 and 509, or any combinations thereof. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) rate of binding for a given nucleotidesubstrate, for example a labeled nucleotide analog, relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits a rate of binding for a particular nucleotide substrate that isat least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%,200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as the rate ofbinding of a reference polymerase for the same nucleotide substrate. Insome embodiments, the reference polymerase is the unmodified counterpartof the modified polymerase. In some embodiments, the referencepolymerase is a Phi-29 polymerase having the amino acid sequence of SEQID NO: 1. In some embodiments, the reference polymerase is a B103polymerase having the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8. In some embodiments, the nucleotide substrate is anatural nucleotide. In some embodiments, the nucleotide substrate is alabeled nucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7 and further includes amino acid mutationsat any one, two, three or more positions selected from the groupconsisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147,166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256,300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389,390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494,497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555,567, 569 and 572, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7. In some embodiments, the modifications caninclude deletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes any one, two, three ormore amino acid mutations selected from the group consisting of: T365G,T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H,H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F,E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y,E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W,A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q,D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S,K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. Optionally, themodified polymerase can further include one or more mutations reducing3′ to 5′ exonuclease activity selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, this modified polymerase comprises the aminoacid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to exhibitan altered branching ratio for one or more nucleotide substrates,particularly for labeled nucleotide analogs. The term “branching” andits variants, when used in reference to a polymerase, typically refersto binding of the appropriate nucleotide to the polymerase withoutproductive incorporation of the nucleotide into an extending nucleicacid molecule by the polymerase. In some applications, for examplesingle molecule sequencing, such behavior can be undesirable because itcan complicate analysis of the reaction as it progresses and/or renderit difficult to distinguish between productive and non-productiveincorporations, and it is therefore desirable to alter the branchingratio to minimize such complications. The complications posed bybranching behavior during single molecule sequencing analysis aredescribed, for example, in Rank, U.S. Published Patent Application No.2008/0108082; Hanzel, U.S. Published Patent Application No.2007/0196846; Clark, U.S. Published Patent Application No. 2009/0176233;and Bjornsen, U.S. Published Patent Application No. 2009/0286245.

Without being bound to any particular theory, in some embodiments, thebranching ratio can be defined as the fraction (expressed as apercentage ratio) of events involving the formation of anenzyme-template-nucleotide ternary complex, E•NA•N—P, that actuallyresult in productive incorporation of the nucleotide into an extendingnucleic acid molecule. Alternatively, in some embodiments, the branchingratio can be defined as the fraction of the nucleotides that bind to,and dissociate from, the polymerase active site without beingincorporated to the extending nucleic acid molecule.

One exemplary mathematical measure for branching ratio can berepresented as follows:Branching ratio=k _(po1)/(k _(po1) +k ⁻¹)

Without intending to be bound to any particular theory, in some casesthis measure of branching ratio, k_(po1)/(k_(po1)+k⁻¹), can provides anindication of the fraction (expressed as a percentage) of nucleotidebinding events (i.e., events wherein the nucleotide binds to thepolymerase to form a ternary enzyme-template-nucleotide complex) thatresult in productive incorporation. In some applications, it can bedesirable to increase the branching ratio as much as possible, ideallyto 1.0 (wherein a value of 1.0 indicates that every bound nucleotidebecomes incorporated into the extending nucleic acid molecule).

In some embodiments, the branching ratio (measured ask_(po1)/(k_(po1)+k⁻¹)) of the modified polymerase for a particularnucleotide substrate, for example, a labeled nucleotide analog, can bebetween about 0.25 and 1.00, typically between about 0.6 and about 0.99.In some embodiments, the branching ratio of the modified polymerase fora particular nucleotide substrate, for example a labeled nucleotideanalog, is greater than 0.95, typically greater than about 0.97, 0.98,0.99 or 0.999.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) branching ratio for a given nucleotidesubstrate, for example a labeled nucleotide analog, relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits a branching ratio for a particular nucleotide substrate that isat least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%,200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as thebranching ratio of a reference polymerase for the same nucleotidesubstrate. In some embodiments, the reference polymerase is theunmodified counterpart of the modified polymerase. In some embodiments,the reference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotide substrateis a natural nucleotide. In some embodiments, the nucleotide substrateis a labeled nucleotide analog. In some embodiments, the modifiedpolymerase comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or any other variant of the polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase comprises one or more amino acid substitutions at positionsselected from the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166,246, 247, 339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384,385, 455, 507 and 509, or any combinations thereof. In some embodiments,the modified polymerase comprises an amino acid sequence that is atleast 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 7 and further includes amino acidmutations at any one, two, three or more positions selected from thegroup consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129,147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252,256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387,389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493,494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552,555, 567, 569 and 572, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the modificationscan include deletions, additions and substitutions. The substitutionscan be conservative or non-conservative substitutions. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 7 and further includes any one,two, three or more amino acid mutations selected from the groupconsisting of: T365G, T365F, T365G, T365S, T365K, T365R, T365A, T365Q,T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R, H370A, H370Q,H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K, E371R, E371A,E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R, K372A,K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A, K380Q,K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R, A481K, A481A,A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T, D507S, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 7. Optionally, the modified polymerase can further include one ormore mutations reducing 3′ to 5′ exonuclease activity selected from thegroup consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F,Y162C, D166A, Q377A and S385G, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) k_(po1)/(k_(po1)+k⁻¹) value for a givennucleotide substrate, for example a labeled nucleotide analog, relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a k_(po1)/(k_(po1)+k⁻¹) value for a particularnucleotide substrate that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% as high as the k_(po1)/(k_(po1)+k⁻¹) value of a referencepolymerase for the same nucleotide substrate. In some embodiments, thereference polymerase is the unmodified counterpart of the modifiedpolymerase. In some embodiments, the reference polymerase is a Phi-29polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the reference polymerase is a B103 polymerase having theamino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the nucleotide substrate is a natural nucleotide. Insome embodiments, the nucleotide substrate is a labeled nucleotideanalog. In some embodiments, the modified polymerase comprises the aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase can be at least 80%, 85%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the modified polymerase comprises one ormore amino acid substitutions at positions selected from the groupconsisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371,372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, orany combinations thereof. In some embodiments, the modified polymerasecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7 and further includes amino acid mutations at any one, two,three or more positions selected from the group consisting of: 2, 9, 12,14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187,195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339,357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511,526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. In some embodiments, the modified polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to exhibiteither increased or decreased processivity for one or more nucleotidesubstrates, particularly for labeled nucleotide analogs. Theprocessivity is the number of nucleotides incorporated for a singlebinding event between the polymerase and the target molecule base-pairedwith the polymerization initiation site.

In some embodiments, the processivity of the modified polymerase withone or more labeled nucleotide analogs can be between about 10 and10,000 nucleotides, typically between about 10 and 100 nucleotides. Forexample, the processivity of the modified polymerase may be about 1, 5,10, 20, 25, 50, 100, 250, 500, 750, 1000, 2000, 5000, or 10,000 or morenucleotides incorporated with a single binding event.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) processivity for a given nucleotide substrate,for example a labeled nucleotide analog, relative to an unmodifiedcounterpart. In some embodiments, the modified polymerase exhibits aprocessivity for a particular nucleotide substrate that is at leastabout 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%,500%, 750%, 1,000%, 5,000% or 10,000% as high as the processivity of areference polymerase for the same nucleotide substrate. In someembodiments, the reference polymerase is the unmodified counterpart ofthe modified polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof.

Without being bound to any particular theory, under some models oneexemplary mathematical expression for processivity can be represented asfollows:Processivity=k _(po1)/(k _(−1(E•DNA)))

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) k_(po1)/(k⁻¹ (E•DNA)) value for a givennucleotide substrate, for example a labeled nucleotide analog, relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a k_(po1)/(k⁻¹ (E•DNA)) value for a particularnucleotide substrate that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% as high as the k_(po1)/(k_(−1 (E•DNA))) value of a referencepolymerase for the same nucleotide substrate. In some embodiments, thereference polymerase is the unmodified counterpart of the modifiedpolymerase. In some embodiments, the reference polymerase is a Phi-29polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the reference polymerase is a B103 polymerase having theamino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the nucleotide substrate is a natural nucleotide. Insome embodiments, the nucleotide substrate is a labeled nucleotideanalog. In some embodiments, the modified polymerase comprises the aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase can be at least 80%, 85%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the modified polymerase comprises one ormore amino acid substitutions at positions selected from the groupconsisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371,372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, orany combinations thereof. In some embodiments, the modified polymerasecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7 and further includes amino acid mutations at any one, two,three or more positions selected from the group consisting of: 2, 9, 12,14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187,195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339,357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511,526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. In some embodiments, the modified polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to extendan increased or decreased fractional extension activity, defined asfraction of nucleic acid templates that are extended by at least onenucleotide in a polymerase reaction under defined reaction conditions.In a typical embodiment, fractional extension efficiency is determinedusing reaction conditions comprising 50 mM Tris, pH 7.5, 50 mM NaCl, 5mM DTT, 100 nM polymerase, 100 nM primer-template duplex, 2 mM MnCl₂ and5 μM of nucleotides at 37° C. (for eukaryotic or bacterial polymerases)or 23° C. (for B103-like and Phi29-like polymerases) for 30 seconds. Insome embodiments, the extended fraction can be measured as thepercentage of total primed nucleic acid templates that are extended byone or more nucleotides under such reaction conditions. In someembodiments, the fractional extension activity of the modifiedpolymerase is increased or decreased relative to the fractionalextension activity of a reference polymerase under identical reactionconditions. In some embodiments, the reference polymerase can be aPhi-29 polymerase having the amino acid sequence of SEQ ID NO: 1. Insome embodiments, the reference polymerase is a B103 polymerase havingthe amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.In some embodiments, the nucleotide substrate is a natural nucleotide.In some embodiments, the nucleotide substrate is a labeled nucleotideanalog. In some embodiments, the modified polymerase comprises the aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase can be at least 80%, 85%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the modified polymerase comprises one ormore amino acid substitutions at positions selected from the groupconsisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371,372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, orany combinations thereof, and exhibits increased or decreased fractionalextension activity as compared to a reference polymerase, e.g., anunmodified counterpart. In some embodiments, the modified polymerasecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7 and further includes amino acid mutations at any one, two,three or more positions selected from the group consisting of: 2, 9, 12,14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187,195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339,357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511,526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. In some embodiments, the modified polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to exhibiteither increased or decreased ease of entry of nucleotides, particularlylabeled nucleotide analogs, into the polymerase active site. In someembodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) degree of steric inhibition of nucleotide entryto the active site relative to the degree of steric inhibition exhibitedby a reference polymerase. In some embodiments, the reference polymeraseis a Phi-29 polymerase having the amino acid sequence of SEQ ID NO: 1.In some embodiments, the reference polymerase is a B103 polymerasehaving the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the nucleotide substrate is a naturalnucleotide. In some embodiments, the nucleotide substrate is a labelednucleotide analog. In some embodiments, the modified polymerasecomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or anyother variant of the polymerase having the amino acid sequence of SEQ IDNO: 7. In some embodiments, the modified polymerase can be at least 80%,85%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 8. In some embodiments, the modified polymerasecomprises one or more amino acid substitutions at positions selectedfrom the group consisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247,339, 370, 371, 372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455,507 and 509, or any combinations thereof. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7 and further includes amino acid mutationsat any one, two, three or more positions selected from the groupconsisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147,166, 176, 185, 186, 187, 195, 208, 221, 246, 247, 248, 251, 252, 256,300, 302, 310, 318, 339, 357, 359, 360, 362, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 380, 383, 384, 385, 386, 387, 389,390, 392, 399, 405, 411, 419, 430, 455, 475, 477, 481, 483, 493, 494,497, 503, 507, 509, 511, 526, 528, 529, 531, 535, 544, 550, 552, 555,567, 569 and 572, wherein the numbering is relative to the amino acidsequence of SEQ ID NO: 7. In some embodiments, the modifications caninclude deletions, additions and substitutions. The substitutions can beconservative or non-conservative substitutions. In some embodiments, themodified polymerase comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7 and further includes any one, two, three ormore amino acid mutations selected from the group consisting of: T365G,T365F, T365G, T365S, T365K, T365R, T365A, T365Q, T365W, T365Y, T365H,H370G, H370T, H370S, H370K, H370R, H370A, H370Q, H370W, H370Y, H370F,E371G, E371H, E371T, E371S, E371K, E371R, E371A, E371Q, E371W, E371Y,E371F, K372G, K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,K372F, K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,A481E, A481F, A481G, A481S, A481R, A481K, A481A, A481T, A481Q, A481W,A481Y, D507H, D507G, D507E, D507T, D507S, D507R, D507A, D507R, D507Q,D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T, K509S,K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 7. Optionally, themodified polymerase can further include one or more mutations reducing3′ to 5′ exonuclease activity selected from the group consisting of:D9A, E11A, E11I, T12I, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A andS385G, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, this modified polymerase comprises the aminoacid substitution H370R.

In some embodiments, the modified polymerase exhibits, or can be furthermodified, selected, mutated, evolved or otherwise engineered to exhibiteither increased or decreased rate of incorporation of one or morenucleotide substrates, particularly for labeled nucleotide analogs. Insome embodiments, the rate of nucleotide incorporation of the modifiedpolymerase with one or more nucleotide substrates is greater than orequal to one nucleotide per second, 5 nucleotides per second, 10nucleotides per second, 20 nucleotides per second, 30 nucleotides persecond, 40 nucleotides per second, 50 nucleotides per second, 100nucleotides, or 200 nucleotides per second. In some embodiments, therate of nucleotide incorporation is one nucleotide per 2, 3, 4, or 5seconds.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) rate of nucleotide incorporation for a givennucleotide substrate, for example a labeled nucleotide analog, relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a rate of nucleotide incorporation for a particularnucleotide substrate that is at least about 5%, 10%, 25%, 37.5%, 50%,75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% as high as the rate of nucleotide incorporation of a referencepolymerase for the same nucleotide substrate. In some embodiments, thereference polymerase is the unmodified counterpart of the modifiedpolymerase. In some embodiments, the reference polymerase is a Phi-29polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the reference polymerase is a B103 polymerase having theamino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the nucleotide substrate is a natural nucleotide. Insome embodiments, the nucleotide substrate is a labeled nucleotideanalog. In some embodiments, the modified polymerase comprises the aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase can be at least 80%, 85%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the modified polymerase comprises one ormore amino acid substitutions at positions selected from the groupconsisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371,372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, orany combinations thereof. In some embodiments, the modified polymerasecomprises an amino acid sequence that is at least 70%, 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7 and further includes amino acid mutations at any one, two,three or more positions selected from the group consisting of: 2, 9, 12,14, 15, 58, 59, 61, 63, 73, 98, 107, 129, 147, 166, 176, 185, 186, 187,195, 208, 221, 246, 247, 248, 251, 252, 256, 300, 302, 310, 318, 339,357, 359, 360, 362, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 380, 383, 384, 385, 386, 387, 389, 390, 392, 399, 405, 411,419, 430, 455, 475, 477, 481, 483, 493, 494, 497, 503, 507, 509, 511,526, 528, 529, 531, 535, 544, 550, 552, 555, 567, 569 and 572, whereinthe numbering is relative to the amino acid sequence of SEQ ID NO: 7. Insome embodiments, the modifications can include deletions, additions andsubstitutions. The substitutions can be conservative or non-conservativesubstitutions. In some embodiments, the modified polymerase comprises anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

In one embodiment, polymerases exhibiting reduced nucleotideincorporation rates include mutant phi29 polymerase having lysinesubstituted with leucine, arginine, histidine or other amino acids(Castro 2009 Nature Structural and Molecular Biology 16:212-218). Insome embodiments, the polymerase can be selected to exhibit eitherreduced or enhanced rates of incorporation for polyphosphate-comprisingnucleotides comprising a label bonded to the terminal phosphate.

In some embodiments, the polymerase can be selected, mutated, modified,evolved or otherwise engineered to exhibit high fidelity with low errorrates. The fidelity of a polymerase may be measured using assays wellknown in the art (Lundburg et al., 1991 Gene, 108:1-6). Typically, thefidelity of a polymerase is measured as the error rate, i.e., thefrequency of incorporation of a nucleotide in a manner that violates thewidely known Watson-Crick base pairing rules. The accuracy or fidelityof DNA polymerization can be influenced not only by the polymeraseactivity of a given enzyme, but also by the 3′-5′ exonuclease activityof a polymerase. The error rate of the polymerase can be one error perabout 100, or about 250, or about 500, or about 1000, or about 1500incorporated nucleotides. High fidelity polymerases include thoseexhibiting error rates of about 5×10⁻⁶ per base pair or lower rates. Bysuitable selection and engineering of the polymerase, the error rate ofthe single-molecule sequencing methods disclosed herein can be furtherimproved. In some embodiments, the polymerase can be further engineeredor modified, e.g., via glycosylation, so as to enhance peptide stabilityand/or performance.

In some embodiments, the selection of the polymerase can be determinedby the level of fidelity desired, such as the error rate per nucleotideincorporation. The frequency of misincorporation (f_(mis)) can bedefined as the ratio between the k_(cat)/K_(d) values for correct andincorrect nucleotides. Fidelity, i.e. the number of correctincorporation events before a mismatch occurs, can be expressed as1/f_(mis).

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) fidelity in the presence of a given nucleotidesubstrate, for example a labeled nucleotide analog, relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits a fidelity in the presence of a particular nucleotide substratethat is at least about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%,150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as thefidelity exhibited by a reference polymerase in the presence of the samenucleotide substrate. In some embodiments, the reference polymerase isthe unmodified counterpart of the modified polymerase. In someembodiments, the reference polymerase is a Phi-29 polymerase having theamino acid sequence of SEQ ID NO: 1. In some embodiments, the referencepolymerase is a B103 polymerase having the amino acid sequence of SEQ IDNO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotidesubstrate is a natural nucleotide. In some embodiments, the nucleotidesubstrate is a labeled nucleotide analog. In some embodiments, themodified polymerase comprises the amino acid sequence of SEQ ID NO: 7,SEQ ID NO: 8, or any other variant of the polymerase having the aminoacid sequence of SEQ ID NO: 7. In some embodiments, the modifiedpolymerase can be at least 80%, 85%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In someembodiments, the modified polymerase comprises one or more amino acidsubstitutions at positions selected from the group consisting of: 2, 9,12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374, 375,376, 377, 380, 383, 384, 385, 455, 507 and 509, or any combinationsthereof. In some embodiments, the modified polymerase comprises an aminoacid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes amino acid mutations at any one, two, three or more positionsselected from the group consisting of: 2, 9, 12, 14, 15, 58, 59, 61, 63,73, 98, 107, 129, 147, 166, 176, 185, 186, 187, 195, 208, 221, 246, 247,248, 251, 252, 256, 300, 302, 310, 318, 339, 357, 359, 360, 362, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 383, 384,385, 386, 387, 389, 390, 392, 399, 405, 411, 419, 430, 455, 475, 477,481, 483, 493, 494, 497, 503, 507, 509, 511, 526, 528, 529, 531, 535,544, 550, 552, 555, 567, 569 and 572, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. In some embodiments, themodifications can include deletions, additions and substitutions. Thesubstitutions can be conservative or non-conservative substitutions. Insome embodiments, the modified polymerase comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7 and furtherincludes any one, two, three or more amino acid mutations selected fromthe group consisting of: T365G, T365F, T365G, T365S, T365K, T365R,T365A, T365Q, T365W, T365Y, T365H, H370G, H370T, H370S, H370K, H370R,H370A, H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E371S, E371K,E371R, E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S,K372R, K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R,K380A, K380Q, K380W, K380Y, K380F, A481E, A481F, A481G, A481S, A481R,A481K, A481A, A481T, A481Q, A481W, A481Y, D507H, D507G, D507E, D507T,D507S, D507R, D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G,K509D, K509R, K509E, K509T, K509S, K509R, K509A, K509Q, K509W, K509Y andK509F, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 7. Optionally, the modified polymerase can further includeone or more mutations reducing 3′ to 5′ exonuclease activity selectedfrom the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D, D63A,Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 7. Optionally, this modifiedpolymerase comprises the amino acid substitution H370R.

Each the kinetic parameters of the modified polymerase, including thevarious parameters described above, can be measured with respect to agiven substrate of the polymerase, including, for example, one or moreparticular nucleotides. In some embodiments, the nucleotide is a labelednucleotide analog, for example, a base-, sugar- or phosphate-labeledanalog. The labeled nucleotide analog can comprise one or more phosphategroups. In some embodiments, the nucleotide analog comprises atriphosphate, tetraphosphate, pentaphosphate, hexaphosphate,heptaphosphate, octaphosphate, nonaphosphate or decaphosphate moieties.The labeled nucleotide analog can comprise an organic dye attached tothe omega or terminal phosphate of the labeled nucleotide analog. Insome embodiments, the organic dye is an Alexa Fluor moiety.

Disclosed herein are modified polymerases exhibiting altered levels oftolerance for the presence of specific labels, particularly inorganiclabels such as nanoparticles. The word “tolerance” and its variants, asused herein with reference to a particular polymerase, refers to theaverage primer extension activity retained by the polymerase in thepresence of a particular label (e.g., dye or nanoparticle) under definedreaction conditions, as compared to the average primer extensionactivity of the polymerase in the absence of the label but underotherwise identical reaction conditions.

In some embodiments, the modified polymerase exhibits increasedtolerance for one or more particular labels as compared to itsunmodified counterpart. Such increased tolerance can be desirablebecause it allows high levels of primer extension activity in reactionscomprising labeled components, such as single molecule sequencingreactions. The labels included in the reaction mixture can optionally beattached to the polymerase, to the nucleotides, and/or to any othercomponent of the reaction mixture. The labels can be organic (e.g.,dyes) or inorganic (e.g., nanoparticles). Typically, the tolerance canvary depending on the reaction conditions, including the type(s) andconcentration of any label(s) present in the reaction mixture. The lowerthe decrease in primer extension activity in the presence of thelabel(s), the greater the tolerance of the polymerase for the particularlabel(s).

The tolerance of a polymerase for a particular label is typicallymeasured by first obtaining the tolerance ratio for the polymerase underdefined reaction conditions. The tolerance ratio is the ratio of theaverage primer extension activity of the polymerase in the presence ofone or more types of label under defined reaction conditions (termed“A_(po1-label)”) and the average primer extension activity of thepolymerase in the absence of the label but under otherwise identicalreaction conditions (termed “A_(po1−)”). This relationship can beexpressed mathematically as follows:Tolerance ratio=A _(po1-label) /A _(po1)

The tolerance is then calculated by converting the tolerance ratio intoa percentage value (%), which can be obtained by multiplying thetolerance ratio, A_(po1-label)/A_(po1), by 100. This relationship can berepresented as follows:Tolerance (%)=(A _(po1-label) /A _(po1))×100

For example, a polymerase that has about 90% tolerance for a particulartype of label will retain on average about 90% of its average primerextension activity in a defined reaction mixture including a particularconcentration of label, where the average primer extension activity ofthe polymerase in the absence of label under otherwise identicalreaction conditions is arbitrarily set as 100%.

In a typical embodiment, tolerance is measured using defined reactionconditions (referred to herein as “standard tolerance assay conditions”)comprising 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM DTT, 100 nM polymerase,100 nM primer-template duplex, 2 mM MnCl₂ and 5 μM of nucleotides, and100 nM of label for 5 minutes at 23° C. The observed primer extensionactivity is measured and compared to the primer extension activity ofthe same polymerase in the absence of any label but under otherwiseidentical reaction conditions (control reaction), which is arbitrarilydefined as having 100% primer extension activity.

Depending on the biological application of interest, however, thetolerance can also be determined under different, i.e., non-standard,assay conditions, for example by measuring the average primer extensionactivity of the polymerase under different reaction conditions includinghigher or lower concentrations of the label, and comparing this activityto the average primer extension activity of the polymerase in theabsence of the label but under otherwise identical reaction conditions.In some embodiments, the reaction conditions can optionally includeexposure to excitation radiation of defined intensity, frequency andduration.

In some embodiments, the label is a nanoparticle and the tolerance ofthe polymerase for the nanoparticle is referred to as the “nanoparticletolerance”. The nanoparticle tolerance of a polymerase can vary with thenature of the nanoparticle label itself.

In one exemplary and non-limiting embodiment, the nanoparticle tolerancecan be measured under standard tolerance assay conditions as follows: apolymerase reaction mixture comprising 50 mM Tris, pH 7.5, 50 mM NaCl, 5mM DTT, 100 nM polymerase, 100 nM primer-template duplex and 2 mM MnCl₂is prepared, as well as a second aliquot of the identical reactionmixture further comprising 100 nM of test nanoparticle. The polymerasereaction is then initiated by the addition of 5 μM of labeled nucleotideto both mixtures, and the resulting primer extension activity ismeasured after 5 minutes. All reactions are performed at 23° C. Thenanoparticle tolerance ratio is calculated by dividing the averageprimer extension activity exhibited by the modified polymerase inreactions comprising 100 nM nanoparticles (A_(po1,np)) by the observedprimer extension activity of the polymerase in control reactions lackingnanoparticles (A_(po1)), as expressed in the following equation:Nanoparticle tolerance ratio=(A _(po1,np) /A _(po1))

The nanoparticle tolerance is then calculated by converting thenanoparticle tolerance ratio into a percentage value, by multiplying thenanoparticle tolerance ratio, A_(po1,np)/A_(po1), by 100. Thisrelationship can be expressed as follows:Nanoparticle tolerance (%)=(A _(po1,np) /A _(po1))×100.

In some embodiments, the present disclosure relates to modifiedpolymerases having a nanoparticle tolerance (A_(po1-label)/A_(po1)×100),of up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 80% or 90% as measured using standard tolerance assayconditions, as described herein. In some embodiments, the nanoparticletolerance of the modified polymerase is at least about 5%, 10%, 25%,50%, 70%, 75%, 80%, 90%, 100%, 125%, 250%, 500%, 750% or 1,000% asmeasured using standard tolerance assay conditions.

In some embodiments, the modified polymerase exhibits an altered, e.g.,increased or decreased, nanoparticle tolerance (A_(po1,np)/A_(po1)×100)relative to an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a photostability (A_(po1,np)/A_(po1)×100) that is atleast about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%,250%, 500%, 750%, 1,000%, 5,000% or 10,000% of the photostability of areference polymerase under identical reaction conditions. In someembodiments, the reaction conditions can comprise standard toleranceassay conditions, as described herein.

In some embodiments, the modified polymerase has a primer extensionactivity (A_(po1-np)) under standard tolerance assay conditions that isat least about that is at least about 5%, 10%, 25%, 37.5%, 50%, 75%,100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% of the primer extension activity of a reference polymerase underidentical reaction conditions.

In some embodiments, the reference polymerase is the unmodifiedcounterpart of the modified polymerase. In some embodiments, thereference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotide substrateis a natural nucleotide. In some embodiments, the nucleotide substrateis a labeled nucleotide analog. In some embodiments, the modifiedpolymerase comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or any other variant of the polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase having altered nanoparticle tolerance comprises one or moreamino acid substitutions at positions selected from the group consistingof: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373,374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, or anycombinations thereof.

In some embodiments the nanoparticle tolerance, expressed as apercentage value (A_(po1,np)/A_(po1)×100) of the modified polymerase canbe about 70%, whereas the A_(po1,np)/A_(po1)×100 value of a referencePhi-29 polymerase comprising the amino acid sequence of SEQ ID NO: 1under identical reaction conditions is about 60%. In some embodimentsthe (A_(po1,np)/A_(po1)×100) value of the modified polymerase can be atleast about 85%, whereas the (A_(po1,np)/A_(po1)×100) value of areference Phi-29 polymerase comprising the amino acid sequence of SEQ IDNO: 1 is no greater than about 55% under identical reaction conditions.In some embodiments the (A_(po1,np)/A_(po1)×100) value of the modifiedpolymerase can be about 90%, whereas the A_(po1,np)/A_(po1)×1000 valueof a reference Phi-29 polymerase comprising the amino acid sequence ofSEQ ID NO: 1 under identical reaction conditions is about 50%.

Disclosed herein are modified polymerases exhibiting altered (e.g.,increased or decreased) levels of photostability following exposure toexcitation radiation. The word “photostability” and its variants, asused herein with reference to a particular polymerase, refers to theaverage primer extension activity retained by the polymerase followingexposure to excitation radiation under defined reaction conditions, ascompared to the average primer extension activity of the polymerase inthe absence of such exposure but under otherwise identical reactionconditions.

In some embodiments, the modified polymerase exhibits increasedphotostability as compared to its unmodified counterpart, thusincreasing its utility in methods and systems involving labels requiringexcitation in order to be detectable. For example, increasedphotostability can be desirable because it allows high levels of primerextension activity in reactions involving the use of excitationradiation, such as single molecule sequencing reactions.

Typically, the photostability will vary depending on the reactionconditions employed, including the intensity, wavelength and duration ofthe excitation radiation. The lower the decrease in primer extensionactivity following exposure to excitation radiation, the greater thephotostability of the polymerase.

The photostability of a polymerase is typically measured by firstobtaining the photostability ratio of the polymerase under definedreaction conditions. The photostability ratio is the ratio of theaverage primer extension activity of the polymerase following exposureto excitation radiation under defined reaction conditions (termed“A_(po1-R)”) and the average primer extension activity of the polymerasewithout exposure to any excitation radiation but under otherwiseidentical reaction conditions (termed “A_(po1-)”). This relationship canbe expressed mathematically as follows:Photostability ratio=A _(po1-R) /A _(po1)

The photostability can then be calculated by converting thephotostability ratio into a percentage value (%), which can be obtainedby multiplying the ratio A_(po1-R)/A_(po1) by 100. This relationship canbe represented as follows:Photostability (%)=(A _(po1-R) /A _(po1))×100

For example, a polymerase that has about 90% photostability will retainon average about 90% of its average primer extension activity underdefined reaction conditions (including exposure to excitation radiationof a defined intensity, wavelength and duration), where the averageprimer extension activity of the polymerase in the absence of anyexposure to excitation radiation but under otherwise identical reactionconditions is arbitrarily set as 100%.

In a typical embodiment, the photostability of a polymerase is measuredusing defined reaction conditions (referred to herein as “standardphotostability assay conditions”) comprising 50 mM Tris, pH 7.5, 50 mMNaCl, 1 mM DTT, 2 mM MnCl₂, 0.3% BSA, 200 nM His-tagged polymerase, 100nM primed template and 100 nM nanoparticles and further includingexposure to excitation radiation of 50 W/cm² intensity at 405 nmwavelength and 5 minutes duration. Following such irradiation, theprimer extension reaction is initiated by adding nucleotides to a finalconcentration of 5 μM and primer extension is performed at 23° C. for 30seconds. The resulting primer extension activity is measured after 30seconds by arresting the reaction (e.g., via addition of 10 mM EDTA).The primer extension activity is then measured and then compared to theprimer extension activity of control sample (i.e., no exposure toexcitation radiation) under otherwise identical reaction conditions.

In one exemplary embodiment, the primer extension activity was measuredby resolving the extension products on a 8M urea, 24% polyacrylamide,and calculating the concentration of fully extended product relative tototal concentration for all extension products. The observed primerextension activity is compared to the primer extension activity of thesame polymerase in the absence of any label but under otherwiseidentical reaction conditions (control reaction), which is arbitrarilydefined as having 100% primer extension activity.

Depending on the biological application of interest, however, thephotostability can also be determined under different, i.e.,non-standard, assay conditions, for example by measuring the averageprimer extension activity of the polymerase under different reactionconditions including higher or lower intensities, wavelengths and/ordurations of excitation, and comparing this activity to the averageprimer extension activity of the polymerase in the absence of the labelbut under otherwise identical reaction conditions. In some embodiments,the reaction conditions can optionally include the presence of one ormore labels, e.g., nanoparticles in the reaction mixture.

In some embodiments, the present disclosure relates to modifiedpolymerases having a photostability (A_(po1-R)/A_(po1)×100) of up toabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 80% or 90% under standard photostability assay conditions, asdescribed herein. In some embodiments, the photostability of themodified polymerase is at least about 5%, 10%, 25%, 50%, 70%, 75%, 80%,90%, 100%, 125%, 250%, 500%, 750% or 1,000% under standardphotostability assay conditions.

In some embodiments, the modified polymerase exhibits an altered, e.g.,increased or decreased, photostability (A_(po1-R)/A_(po1)×100) relativeto an unmodified counterpart. In some embodiments, the modifiedpolymerase exhibits a photostability (A_(po1-R)/A_(po1)×100) that is atleast about 5%, 10%, 25%, 37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%,250%, 500%, 750%, 1,000%, 5,000% or 10,000% as high as thephotostability of a reference polymerase under identical reactionconditions. In some embodiments, the reaction conditions can comprisestandard photostability assay conditions, as described herein.

In some embodiments, the modified polymerase has a primer extensionactivity (A_(po1-R)) under standard photostability assay conditions thatis at least about that is at least about 5%, 10%, 25%, 37.5%, 50%, 75%,100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%, 5,000% or10,000% of the primer extension activity of a reference polymerase underidentical reaction conditions.

In some embodiments, the reference polymerase is the unmodifiedcounterpart of the modified polymerase. In some embodiments, thereference polymerase is a Phi-29 polymerase having the amino acidsequence of SEQ ID NO: 1. In some embodiments, the reference polymeraseis a B103 polymerase having the amino acid sequence of SEQ ID NO: 6, SEQID NO: 7 or SEQ ID NO: 8. In some embodiments, the nucleotide substrateis a natural nucleotide. In some embodiments, the nucleotide substrateis a labeled nucleotide analog. In some embodiments, the modifiedpolymerase comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, or any other variant of the polymerase having the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the modified polymerase can be atleast 80%, 85%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the modifiedpolymerase having altered photostability comprises one or more aminoacid substitutions at positions selected from the group consisting of:2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371, 372, 373, 374,375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, or any combinationsthereof.

In some embodiments, the excitation radiation comprises light of 405 nmwavelength. The intensity of excitation radiation can be about 10 W/cm²,20 W/cm² or 50 W/cm² or any other suitable value. In some embodiments,the duration of exposure to excitation radiation can be less than orequal to 0.1, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 2.5, 5, 10, 15, 20, 30, 45or 60 minutes. In some embodiments, the A_(po1-R)/A_(po1) value of themodified polymerase is at least 50%, 60%, 70%, 80%, 90%, 95% or 99%following exposure to excitation radiation at 20 W/cm² for 5 minutes. Insome embodiments, the A_(po1-R)/A_(po1) value of the modified polymeraseis at least 50%, 60%, 70%, 80%, 90%, 95% or 99% following exposure toexcitation radiation at 50 W/cm² for 30 seconds.

In some embodiments, the modified polymerase exhibits an altered (e.g.,increased or decreased) A_(po1-R)/A_(po1) value relative to anunmodified counterpart. In some embodiments, the modified polymeraseexhibits A_(po1-R)/A_(po1) value that is at least about 5%, 10%, 25%,37.5%, 50%, 75%, 100%, 110%, 125%, 150%, 200%, 250%, 500%, 750%, 1,000%,5,000% or 10,000% as high as the A_(po1-R)/A_(po1) value of a referencepolymerase under identical reaction conditions. In some embodiments, thereference polymerase is the unmodified counterpart of the modifiedpolymerase. In some embodiments, the reference polymerase is a Phi-29polymerase having the amino acid sequence of SEQ ID NO: 1. In someembodiments, the reference polymerase is a B103 polymerase having theamino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the nucleotide substrate is a natural nucleotide. Insome embodiments, the nucleotide substrate is a labeled nucleotideanalog. In some embodiments, the modified polymerase comprises the aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or any other variant of thepolymerase having the amino acid sequence of SEQ ID NO: 7. In someembodiments, the modified polymerase can be at least 80%, 85%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ IDNO: 8. In some embodiments, the modified polymerase comprises one ormore amino acid substitutions at positions selected from the groupconsisting of: 2, 9, 12, 58, 59, 63, 129, 166, 246, 247, 339, 370, 371,372, 373, 374, 375, 376, 377, 380, 383, 384, 385, 455, 507 and 509, orany combinations thereof.

Also disclosed herein are kits comprising one or more modifiedpolymerases of the present disclosure for use in primer extension orsingle molecule sequencing reactions. For example, in some embodiments,the kit can comprise one or more modified polymerases and one or morenucleotides packaged in a fashion to enable use of the polymerase toincorporate one or more nucleotides of the kit. The modified polymerasecan be a polymerase having or comprising the amino acid sequence of SEQID NO: 7 or SEQ ID NO: 8, or a polymerase comprising an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. Thenucleotide can be a labeled nucleotide analog. The label can be linkedto the sugar, base, phosphate or any other moiety of the nucleotideanalog. In some embodiments, the nucleotide is a reversible terminator.

In some embodiments, the kit can comprise a modified polymerasesaccording to the present disclosure linked to one or more labels to forma labeled polymerase conjugates. Some methods of making and usinglabeled polymerase conjugates are disclosed, for example, in U.S.provisional application Nos. 61/184,770, filed Jun. 5, 2009; 61/245,457,filed on Sep. 24, 2009; and 61/299,919, filed on Jan. 29, 2010, as wellas U.S. application Ser. No. 12/748,355 titled “Conjugates ofBiomolecules to Nanoparticles” and filed Mar. 26, 2010; and U.S.application Ser. No. 12/748,314 titled “Labeled Enzyme Compositions,Methods & Systems” and filed Mar. 26, 2010. In some embodiments, the oneor more labels of the labeled polymerase conjugate are capable ofundergoing FRET with the label of the nucleotide bound to the activesite of the polymerase. Optionally, such FRET can occur with a FRETefficiency of at least about 10% or 20%.

In some embodiments, the kit can further comprise additional reagents,such as additional nucleotides, including labeled and unlabelednucleotides; divalent metal cations; nucleic acid molecules (forexample, for use as primer and/or template); buffer solutions; saltsolutions, and the like. Typically, the kit will also compriseinstructions for use of the contents in a variety of applications suchas, for example, nucleotide incorporation, primer extension and singlemolecule sequencing.

Also provided herein are methods of isolating or preparing the modifiedpolymerases of the present disclosure. In some embodiments, thepolymerase can be a recombinant protein which is produced by a suitableexpression vector/host cell system. The polymerases can be encoded bysuitable recombinant expression vectors carrying inserted sequences ofthe polymerases. The polymerase sequence can be linked to a suitableexpression vector. The polymerase sequence can be inserted in-frame intothe suitable expression vector. The suitable expression vector canreplicate in a phage host, or a prokaryotic or eukaryotic host cell. Thesuitable expression vector can replicate autonomously in the host cell,or can be inserted into the host cell's genome and be replicated as partof the host genome. The suitable expression vector can carry aselectable marker that confers resistance to drugs (e.g., kanamycin,ampicillin, tetracycline, chloramphenicol, or the like) or requirementfor nutrients. The suitable expression vector can have one or morerestriction sites for inserting the nucleic acid molecule of interest.The suitable expression vector can include expression control sequencesfor regulating transcription and/or translation of the encoded sequence.The expression control sequences can include: promoters (e.g., inducibleor constitutive), enhancers, transcription terminators, and secretionsignals. The expression vector can be a plasmid, cosmid, or phagevector. The expression vector can enter a host cell which can replicatethe vector, produce an RNA transcript of the inserted sequence, and/orproduce protein encoded by the inserted sequence. Methods for preparingsuitable recombinant expression vectors and expressing the RNA and/orprotein encoded by the inserted sequences are well known. See, e.g.,Sambrook et al., Molecular Cloning (1989).

As the skilled artisan will readily appreciate, the scope of the presentdisclosure encompasses not only the specific amino acid and/ornucleotide sequences disclosed herein, but also, for example, too manyrelated sequences encoding genes and/or peptides with the functionalproperties described herein. For example, nucleotide and amino acidsequences encoding conservative variants of the various modifiedpolymerases disclosed herein are also within the scope of the presentdisclosure.

Also provided herein are methods of using the modified polymerasecompositions of the present disclosure.

In some embodiments, the modified polymerases of the present disclosurecan polymerize one or more nucleotides, including, for example, labeledand unlabeled nucleotides.

In some embodiments, the disclosure relates to a method for performing aprimer extension reaction, comprising: contacting a modified DNApolymerase as provided herein with a nucleic acid molecule and anucleotide under conditions where the nucleotide is incorporated intothe nucleic acid molecule by the modified DNA polymerase. Optionally,the modified DNA polymerase comprises an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 7.

Optionally, the nucleotide is a labeled nucleotide, and the label of thenucleotide emits a signal during incorporation of the at least onenucleotide. Optionally, the method further comprises detecting thesignal emitted by the nucleotide label. Optionally, the method furthercomprises analyzing the detected signal to determine the identity of theincorporated nucleotide.

Optionally, the modified DNA polymerase can exhibit a t⁻¹ value for alabeled nucleotide that is equal to or greater than the t⁻¹ value of areference Phi-29 polymerase comprising the amino acid sequence of SEQ IDNO: 1 for the same nucleotide. Optionally, the modified polymeraseexhibits a t_(po1) value for a labeled nucleotide that is equal to orgreater than the t_(po1) value of a reference Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 1 for the samenucleotide. Optionally, the modified DNA polymerase exhibits a residencetime for a labeled nucleotide that is equal to or greater than theresidence time of a reference Phi-29 polymerase comprising the aminoacid sequence of SEQ ID NO: 1 for the same nucleotide. In someembodiments, the modified DNA polymerase has a photostability of atleast about 80% under standard photostability assay conditions. In someembodiments, the modified polymerase has a nanoparticle tolerance of atleast about 80% under standard tolerance assay conditions.

Optionally, the nucleotide is a labeled nucleotide. In some embodiments,the labeled nucleotide comprises a nucleotide label linked to the base,sugar or phosphate group of the nucleotide. In some embodiments, thelabeled nucleotide is a reversible terminator. Optionally, the modifiedDNA polymerase can be linked to a label. The label can be a dye or ananoparticle. Optionally, the label of the modified polymerase and thenucleotide label are capable of undergoing FRET with each other. SuchFRET can optionally occur with a FRET efficiency of at least 20%.

Disclosed herein are methods for incorporation of one or morenucleotides onto the end of a nucleic acid molecule, comprising:contacting a nucleic acid molecule with a modified polymerase providedfor herein and one or more nucleotides under conditions where at leastone nucleotide is incorporated by the modified polymerase. The nucleicacid molecule can be any suitable target nucleic acid molecule ofinterest. In some embodiments, the modified polymerase can be apolymerase having or comprising the amino acid sequence of SEQ ID NO: 7or SEQ ID NO: 8. In some embodiments, the at least one nucleotide can bebecome incorporated onto the 3′ end of an extending nucleic acidmolecule by the polymerase. In some embodiments, the at least onenucleotide can be a labeled nucleotide analog. The labeled nucleotideanalog can comprise a label linked to the base, sugar, phosphate or anyother portion of the nucleotide analog. In some embodiments, thenucleotide can also comprise a blocking group that inhibits, slows downor blocks further incorporation of nucleotides onto the end of thenucleic acid molecule until the blocking group is removed from thenucleotide. In some embodiments, the nucleotide comprising a blockinggroup is a reversible terminator for nucleic acid synthesis, asdescribed further below. In some embodiments, the blocking group can beremoved from the nucleotide by chemical, enzymatic, or photocleavingreactions.

In some embodiments, the method further includes the step of adding oneor more divalent cations to the polymerase reaction mixture in an amountsufficient for inhibiting further incorporation of nucleotides onto theend of the nucleic acid molecule by the modified polymerase. In someembodiments, the divalent cation that inhibits nucleotide incorporationis calcium. In another embodiment, omitting, reducing, or chelatingcations that permit nucleotide incorporation (e.g, manganese and/ormagnesium) can be employed. Such methods are described, for example, inU.S. Provisional Application 61/242,762, filed Sep. 15, 2009; and inU.S. Provisional Application No. 61/184,774, filed on Jun. 5, 2009. Insome embodiments, the polymerase can be linked to a label, as, forexample, disclosed in U.S. Provisional Application No. 61/184,770, filedJun. 5, 2009.

Also provided herein is a method for detecting one or more nucleotideincorporations onto the end of a single nucleic acid molecule,comprising: contacting a target nucleic acid molecule with a modifiedpolymerase provided for herein and one or more labeled nucleotides underconditions where at least one labeled nucleotide is incorporated by thepolymerase onto the end of an extending nucleic acid molecule and wherethe label of at least one labeled nucleotide emits one or more signalsindicative of nucleotide incorporation; and detecting the one or moresignals indicative of nucleotide incorporation. In some embodiments, themethod can further include the step of analyzing the one or moredetected signals indicative of nucleotide incorporation to determine thepresence of a target nucleic acid molecule. In some embodiments, thedetecting can be performed in real or near real time. In someembodiments, the method can further include the step of analyzing theone or more detected signals indicative of nucleotide incorporation todetermine the identity of the target nucleic acid molecule. In someembodiments, the method can further include the step of analyzing theone or more detected signals indicative of nucleotide incorporation todetermine the identity of one or more incorporated nucleotides. In someembodiments, a time series of nucleotide incorporations can be detectedand analyzed to determine some or all of the sequence of the targetnucleic acid molecule.

Also disclosed herein is a method for determining a nucleotide sequenceof a single nucleic acid molecule, comprising: (a) conducting apolymerase reaction comprising a modified polymerase provided for hereinand at least one labeled nucleotides, which reaction results in theincorporation of one or more labeled nucleotides by the polymerase andthe generation of one or more signals indicative of one or morenucleotide incorporations; (b) detecting a time sequence of nucleotideincorporations; and (c) determining the identity of one or moreincorporated nucleotides, thereby determining some or all of thenucleotide sequence of a single nucleic acid molecule.

Also provided herein are methods of sequencing a nucleic acid molecule,comprising: (a) conducting a polymerase reaction comprising a modifiedpolymerase and at least one labeled nucleotide, which reaction resultsin the incorporation of one or more labeled nucleotides by thepolymerase and the generation of one or more signals indicative of oneor more nucleotide incorporations; (b) detecting a time sequence ofnucleotide incorporations; and (c) determining the identity of one ormore incorporated nucleotides, thereby determining some or all of thenucleotide sequence of a single nucleic acid molecule.

In some embodiments, the polymerase can bind a target nucleic acidmolecule, which may or may not be base-paired with a polymerizationinitiation site (e.g., primer).

The polymerization initiation site is used by the polymerase (e.g., DNAor RNA polymerase) to initiate nucleotide polymerization. In someembodiments, the polymerization initiation site can be a terminal 3′ OHgroup. The 3′ OH group can serve as a substrate for the polymerase fornucleotide polymerization. The 3′ OH group can serve as a substrate forthe polymerase to form a phosphodiester bond between the terminal 3′ OHgroup and an incorporated nucleotide. The 3′ OH group can be providedby: the terminal end of a primer molecule; a nick or gap within anucleic acid molecule (e.g., oligonucleotide) which is base-paired withthe target molecule; the terminal end of a secondary structure (e.g.,the end of a hairpin-like structure); or an origin of replication. Thepolymerization initiation site can be provided by an accessory protein(e.g., RNA polymerase or helicase/primase). The polymerizationinitiation site can be provided by a terminal protein which can be bound(covalently or non-covalently) to the end of the target nucleic,including terminal protein (e.g., TP) found in phage (e.g., TP fromphi29 phage). Thus, the polymerization initiation site may be at aterminal end or within a base-paired nucleic acid molecule.

In other embodiments, the polymerization initiation site used by somepolymerases (e.g., RNA polymerase) may not include a 3′ OH group.

The portion of the target molecule which is base paired with the primeror with the oligonucleotide, or the self-primed portion of the targetmolecule, can form hydrogen bonding by Watson-Crick or Hoogstein bindingto form a duplex nucleic acid structure. The primer, oligonucleotide,and self-priming sequence may be complementary, or partiallycomplementary, to the nucleotide sequence of the target molecule. Thecomplementary base pairing can be the standard A-T or C-G base pairing,or can be other forms of base-pairing interactions.

The polymerization initiation site can be in a position on the targetnucleic acid molecule to permit successive nucleotide incorporationevents in a direction away from, or towards, the solid surface.

The primer molecule can hybridize with the target nucleic acid molecule.The sequence of the primer molecule can be complementary ornon-complementary with the sequence of the sequence of the targetmolecule. The 3′ terminal end of the primer molecule can provide thepolymerization initiation site.

The primers can be modified with a chemical moiety to protect the primerfrom serving as a polymerization initiation site or as a restrictionenzyme recognition site. The chemical moiety can be a natural orsynthetic amino acid linked through an amide bond to the primer.

The primer, oligonucleotide, or self-priming portion, may benaturally-occurring, or may be produced using enzymatic or chemicalsynthesis methods. The primer, oligonucleotide, or self-priming portionmay be any suitable length including 5, 10, 15, 20, 25, 30, 40, 50, 75,or 100 nucleotides or longer in length. The primer, oligonucleotide, orself-priming portion may be linked to an energy transfer moiety (e.g.,donor or acceptor) or to a reporter moiety (e.g., a dye) using methodswell known in the art.

The primer molecule, oligonucleotide, and self-priming portion of thetarget molecule, may comprise ribonucleotides, deoxyribonucleotides,ribonucleotides, deoxyribonucleotides, peptide nucleotides, modifiedphosphate-sugar backbone nucleotides including phosphorothioate andphosphoramidate, metallonucleosides, phosphonate nucleosides, and anyvariants thereof, or combinations thereof.

In one embodiment, the primer molecule can be a recombinant DNAmolecule. The primer can be linked at the 5′ or 3′ end, or internally,with at least one binding partner, such as biotin. The biotin can beused to immobilize the primer molecule to the surface (via anavidin-like molecule), or for attachment to a reporter moiety. Theprimer can be linked to at least one energy transfer moiety, such as afluorescent dye or a nanoparticle, or to a reporter moiety. The primermolecule can hybridize to the target nucleic acid molecule. The primermolecule can be used as a capture probe to immobilize the targetmolecule.

Typically, the polymerase can selectively bind to a nucleotide. Suchnucleotide binding can occur in a template-dependent ornon-template-dependent manner. Typically, the polymerase can mediatecleavage of the bound nucleotide. Typically, such cleavage of thenucleotide results in the formation of at least two nucleotide cleavageproducts. For polyphosphate-comprising nucleotides, such cleavage willtypically occur between the α and β phosphate groups. Typically, thepolymerase can mediate incorporation of one of the nucleotide cleavageproducts into a nucleic acid molecule, and release of another nucleotidecleavage product. When used in conjunction with polyphosphate-comprisingnucleotides, the released nucleotide cleavage product can comprise oneor more phosphates (for example, a polyphosphate chain); for nucleotidesthat are non-phosphate-comprising analogs, the nucleotide cleavageproduct may not comprise any phosphorus.

In some embodiments, the polymerase can mediate incorporation of anucleotide on to a polymerization initiation site (e.g., terminal 3′OHof a primer).

The compositions, methods, systems and kits of the present disclosurehave particular use in single molecule sequencing reactions. Typically,such applications comprise the performance of a polymerase reactionusing the α conjugate comprising a polymerase linked to a label andhaving polymerase activity according to the present disclosure.

In one exemplary embodiment, the temporal order of nucleotideincorporations during the polymerase reaction is detected and monitoredin real time based on detection of FRET signals resulting from FRETbetween the labeled polymerase conjugates and the nucleotide label of anincorporating acceptor-labeled nucleotide.

In some embodiments, the polymerase is linked to a FRET donor andcontacted with a nucleotide comprising a FRET acceptor. In someembodiments, the donor performs FRET with the acceptor when thepolymerase and nucleotide are bought into sufficient proximity (forexample, during a productive incorporation, a non-productiveincorporation or during association of a nucleotide with the polymeraseactive site), resulting in the emission of a FRET signal. The FRETsignal can optionally be detected and analyzed to determine theoccurrence of a polymerase-nucleotide interaction.

In some embodiments, the FRET can occur prior to, during or afterproductive incorporation of the nucleotide into a nucleic acid molecule.Alternatively, the FRET can occur prior to binding of the nucleotide tothe polymerase active site, or while the nucleotide resides within thepolymerase active site, during a non-productive incorporation.

In some embodiments, the FRET acceptor moiety can in some embodiments beattached to, or comprise part of, the nucleotide sugar, the nucleobase,or analogs thereof. In some embodiments, the FRET acceptor is attachedto a phosphate group of the nucleotide that is cleaved and released uponincorporation of the underlying nucleotide into the primer strand, forexample the γ-phosphate, the β-phosphate or some other terminalphosphate of the incoming nucleotide. When this acceptor-labelednucleotide polyphosphate is incorporated by the labeled polymeraseconjugate into a nucleic acid molecule, the polymerase cleaves the bondbetween the alpha and beta phosphate, thereby releasing a pyrophosphatemoiety comprising the acceptor that diffuses away. Thus, in theseembodiments, a signal indicative of nucleotide incorporation isgenerated through FRET between the nanoparticle and the acceptor bondedto the gamma, beta or other terminal phosphate as each incomingnucleotide is incorporated into the newly synthesized strand. Byreleasing the label upon incorporation, successive incorporation oflabeled nucleotides can each be detected without interference fromnucleotides previously incorporated into the complementary strand.Alternatively, the nucleotide may be labeled with a FRET acceptor moietyon an internal phosphate, for example, the alpha phosphate, the betaphosphate, or another internal phosphate. Although such alpha-phosphateadducts are not cleaved and released during the polymerization process,they can be removed and/or rendered inoperable through appropriatetreatments, e.g., chemical cleavage or photobleaching, later in thesequencing process.

The polymerase reaction conditions can comprise any suitable reactionconditions that permit nucleotide polymerization by labeled polymeraseconjugates of the present disclosure. In one non-limiting example ofnucleotide polymerization, the steps of polymerization can comprise: (1)complementary base-pairing of a target DNA molecule (e.g., a templatemolecule) with a primer molecule having a terminal 3′ OH (the terminal3′ OH provides the polymerization initiation site for the polymerase);(2) binding of the polymerase of the conjugate to the base-paired targetDNA/primer duplex to form a complex (e.g., open complex); (3) binding ofthe candidate nucleotide by the polymerase of the conjugate, whichpolymerase interrogates the candidate nucleotide for complementaritywith the template nucleotide on the target DNA molecule; (4) catalysisof nucleotide polymerization by the polymerase of the conjugate.

In one embodiment, the polymerase of the conjugate comprises cleavage ofthe incorporating nucleotide by the polymerase, accompanied byliberation of a nucleotide cleavage product. When the nucleotide is aphosphate-comprising nucleotide, the cleavage product can include one ormore phosphate groups. In other embodiments, where the polymeraseincorporates a nucleotide analog having substituted phosphate groups,the cleavage product may include one or more substituted phosphategroups.

The candidate nucleotide may or may not be complementary to the templatenucleotide on the target molecule. The candidate nucleotide maydissociate from the polymerase. If the candidate nucleotide dissociatesfrom the polymerase, it can be liberated; in some embodiments, theliberated nucleotide carries intact polyphosphate groups. When thecandidate nucleotide dissociates from the DNA polymerase, the event isknown as a “non-productive binding” event. The dissociating nucleotidemay or may not be complementary to the template nucleotide on the targetmolecule.

The incorporated nucleotide may or may not be complementary to thetemplate nucleotide on the target. When the candidate nucleotide bindsthe DNA polymerase and is incorporated, the event is a “productivebinding” event. The incorporated nucleotide may or may not becomplementary to the template nucleotide on the target molecule.

The length of time, frequency, or duration of the binding of thecomplementary candidate nucleotide to the polymerase can differ fromthat of the non-complementary candidate nucleotide. This time differencecan be used to distinguish between the complementary andnon-complementary nucleotides, and/or can be used to identify theincorporated nucleotide, and/or can be used to deduce the sequence ofthe target molecule.

The signal (or change in signal) generated by the energy transfer donorand/or acceptor can be detected before, during, and/or after anynucleotide incorporation event.

In some embodiments, the polymerase reaction includes RNA polymerizationwhich does not require a 3′ polymerization initiation site. Polymerasereactions involving RNA polymerization are well known in the art.

Productive and Non-Productive Binding

Also provided herein are energy transfer compositions and methods fordistinguishing between the productive and non-productive binding events.The compositions and methods can also provide base identity informationduring nucleotide incorporation. The compositions include nucleotidesand polymerases each attached to a energy transfer moiety.

The compositions and methods provided herein can be used to distinguishevents such as productive and non-productive nucleotide binding to thepolymerase. In a productive binding event, the nucleotide canbind/associate with the polymerase for a time period which isdistinguishable (e.g., longer or shorter time period), compared to anon-productive binding event. In a non-productive binding event, thenucleotide can bind/associate with the polymerase and then dissociate.The donor and acceptor energy transfer moieties produce detectablesignals when they are in proximity to each other and can be associatedwith productive and non-productive binding events. Thus, the time-lengthdifference between signals from the productive and non-productivebinding events can provide distinction between the two types of events.

The detectable signals can be classified into true positive and falsepositive signals. For example, the true positive signals can arise fromproductive binding in which the nucleotide binds the polymerase and isincorporated. The incorporated nucleotide can be complementary to thetemplate nucleotide. In another example, the false positive signals canarise from different binding events, including: non-specific binding,non-productive binding, and any event which brings the energy transferdonor and acceptor into sufficient proximity to induce a detectablesignal.

Optionally, polymerase reactions performed using the methods, systems,compositions and kits of the present disclosure can be performed underany conditions which are suitable for: forming the complex(target/polymerase or target/initiation site/polymerase); binding thenucleotide to the polymerase; permitting the energy transfer andreporter moieties to generate detectable signals when the nucleotidebinds the polymerase; incorporating the nucleotide; permitting theenergy transfer and reporter moieties to generate a signal upon closeproximity and/or nucleotide incorporation; and/or detecting the signal,or change in the signal, from the energy transfer or reporter moieties.The suitable conditions include well known parameters for time,temperature, pH, reagents, buffers, reagents, salts, co-factors,nucleotides, target DNA, primer DNA, enzymes such as nucleicacid-dependent polymerase, amounts and/or ratios of the components inthe reactions, and the like. The reagents or buffers can include asource of monovalent ions, such as KCl, K-acetate, NH₄-acetate,K-glutamate, NH₄Cl, or ammonium sulfate. The reagents or buffers caninclude a source of divalent ions, such as Mg²⁺ and/or Mn²⁺, MgCl₂, orMg-acetate. The buffer can include Tris, Tricine, HEPES, MOPS, ACES, orMES, which can provide a pH range of about 5.0 to about 9.5. The buffercan include chelating agents such as EDTA and EGTA, and the like.

Reducing Photo-Damage

The suitable polymerase reaction conditions can also include compoundswhich reduce photo-damage. For example, the compounds may reduceoxygen-damage or photo-damage. Illuminating the nucleotide bindingand/or nucleotide incorporation reactions with electromagnetic radiationat an excitation wavelength can induce formation of reactive oxygenspecies from the fluorophore or other components in the reaction. Thereactive oxygen species can cause photo-damage to the fluorophores,polymerases, or any other component of the binding or incorporationreactions. The nucleotide binding or nucleotide incorporation reactionscan include compounds which are capable of reducing photo-damage,including: protocatechuate-3,4-dioxygenase, protocatechuic acid;6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (TROLOX); orcyclooctatetraene (COT).

Other compounds for reducing photo-damage include: ascorbic acid,astazanthin, bilirubin, biliverdin, bixin, captopril, canthazanthin,carotene (alpha, beta, and gamma), cysteine, beta-dimethyl cysteine,N-acetyl cysteine, diazobicyclooctane (DABCO), dithiothreitol (DTT),ergothioneine, glucose oxidase/catalase (GO/Cat), glutathione,glutathione peroxidase, hydrazine (N₂H₄), hydroxylamine, lycopene,lutein, polyene dialdehydes, melatonin, methionine,mercaptopropionylglycine, 2-mercaptoethane sulfonate (MESNA), pyridoxine1 and its derivatives, mercaptoethylamine (MEA), β-mercaptoethanol(BME), n-propyl gallate, p-phenylenediamene (PPD), hydroquinone, sodiumazide (NaN₃), sodium sulfite (Na₂SO₃), superoxide dismutase,tocopherols, α-tocopheryl succinate and its analogs, and zeaxanthin.

Also provided herein are methods of using the labeled biomoleculeconjugates of the present disclosure.

For example, disclosed herein are methods for incorporation of one ormore nucleotides onto the end of a nucleic acid molecule, comprising:contacting a conjugate including a polymerase linked to a label with anucleotide under conditions where the nucleotide is incorporated into anucleic acid molecule by the conjugate. The nucleic acid molecule can beany suitable target nucleic acid molecule of interest. In someembodiments, the labeled polymerase can be a polymerase having orcomprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. Insome embodiments, the nucleotide can be become incorporated onto the 3′end of an extending nucleic acid molecule by the polymerase. In someembodiments, the nucleotide can be a labeled nucleotide analog. Thelabeled nucleotide analog can further comprise a label linked to thebase, sugar, phosphate or any other portion of the nucleotide analog. Insome embodiments, the nucleotide can also comprise a blocking group thatinhibits, slows down or blocks further incorporation of nucleotides ontothe end of the nucleic acid molecule until the blocking group is removedfrom the nucleotide. In some embodiments, the nucleotide comprising ablocking group is a reversible terminator for nucleic acid synthesis, asdescribed further below. In some embodiments, the blocking group can beremoved from the nucleotide by chemical, enzymatic, or photocleavingreactions.

In some embodiments, the method further includes the step of adding oneor more divalent cations to the polymerase reaction mixture in an amountsufficient for inhibiting further incorporation of nucleotides onto theend of the nucleic acid molecule by the labeled polymerase. In someembodiments, the divalent cation that inhibits nucleotide incorporationis calcium. In another embodiment, omitting, reducing, or chelatingcations that permit nucleotide incorporation (e.g, manganese and/ormagnesium) can be employed. Such methods are described, for example, inU.S. Provisional Application 61/242,762, filed Sep. 15, 2009; and inU.S. Provisional Application No. 61/184,774, filed on Jun. 5, 2009. Insome embodiments, the polymerase can be linked to a label, as, forexample, disclosed herein and in U.S. Provisional Application No.61/184,770, filed Jun. 5, 2009.

Also provided herein is a method for detecting one or more nucleotideincorporations, comprising: contacting a conjugate including apolymerase linked to a label with a labeled nucleotide under conditionswhere the labeled nucleotide is incorporated by the conjugate into anucleic acid molecule, and where the label of the labeled nucleotideemits a signal indicative of such nucleotide incorporation; anddetecting the signal indicative of such nucleotide incorporation. Insome embodiments, the detecting can be performed in real or near realtime. In some embodiments, the method can further include analyzing thedetected signal indicative of nucleotide incorporation to determine theidentity of the incorporated nucleotide. In some embodiments, thelabeled polymerase conjugate catalyzes a time series of nucleotideincorporations, which can collectively be detected and analyzed todetermine some or all of the sequence of the target nucleic acidmolecule.

Also disclosed herein is a method for determining a nucleotide sequenceof a single nucleic acid molecule, comprising: (a) conducting apolymerase reaction comprising a labeled biomolecule conjugate and alabeled nucleotide under conditions where the conjugate incorporates thelabeled nucleotide into a nucleic acid molecule and a signal indicativeof such nucleotide incorporation is generated; (b) detecting the signalindicative of such nucleotide incorporation; and (c) analyzing thesignal to determine the identity of the incorporated nucleotide.Optionally, a time series of nucleotide incorporation signals can bedetected and analyzed, thereby determining some or all of the nucleotidesequence of a single nucleic acid molecule.

Also provided herein are methods of sequencing a nucleic acid molecule,comprising: (a) performing a polymerase reaction comprising a labeledpolymerase conjugate and labeled nucleotides under conditions resultingin a series of labeled nucleotide incorporations by the polymerase andthe generation of a signal indicative of each nucleotide incorporationthe series; (b) detecting a time sequence of nucleotide incorporations;and (c) determining the identity of one or more incorporatednucleotides, thereby determining some or all of the nucleotide sequenceof a single nucleic acid molecule.

In some embodiments, the polymerase is attached to or associated with asubstrate or surface. In some embodiments, the polymerase can beattached to or associated with a nucleic acid molecule (termed atemplate), and polymerize one or more nucleotides in atemplate-dependent fashion. In some embodiments, the template can beattached to or associated with a substrate or surface. In someembodiments, the polymerase, template, nucleotide, substrate or surface,or some combination thereof, can also be labeled.

In some embodiments, the methods of the present disclosure can beperformed in multiplex and/or “high-throughput” format wherein multipleunits of the labeled polymerase conjugates of the present disclosure caneach be visualized and monitored in parallel with each other. Forexample, in some embodiments, multiple labeled polymerase conjugates maybe positioned, associated with, or attached to different locations on asubstrate, and a polymerase activity of one or more of these polymerasesmay be detected in isolation. In some embodiments, the polymerase or thetemplate nucleic acid molecule are associated with or attached to asubstrate or surface in array format. The array can be spatiallyaddressable.

In some embodiments, the sequencing reaction can be performed usingbuffer conditions comprising 50 mM Tris buffer pH 7.5, 50 mM NaCl, 0-10mM MgCl₂, 2 mM MnCl₂, 330 nM polymerase, 100 nM primed template and 4 μMlabeled nucleotide hexaphosphate. Optionally, 0.3% BSA and/or 0.05%Tween20 can be included in the reaction mix. In some embodiments, thereaction mix is further supplemented with 2 mM DTT and/or singlestranded binding protein (SSBP) at a concentration of 100 μg/ml.

Alternatively, in some embodiments the sequencing reaction can beperformed using buffer conditions comprising 50 mM Tris pH 8.0, 50 mMNaCl and 10 mM MgCl₂.

In one exemplary embodiment, a nucleic acid sequencing system cancomprise a template nucleic acid molecule attached to a substrate, alabeled polymerase conjugate comprising a FRET donor label linked to apolymerase, and labeled nucleotides each comprising a nucleotide linkedto one or more FRET acceptor labels.

The template nucleic acid molecule of this sequencing system can beattached to any suitable substrate or surface using any suitable method.in some embodiments, the template nucleic acid molecule can comprise oneor more biotin moieties, the surface can comprise an avidin moiety, andthe template nucleic acid is linked to the surface via one or morebiotin-avidin bonds. In some embodiments, the template and surface caneach comprise one or more biotin moieties, and be linked to each otherthrough a linkage comprising an avidin moiety.

In some embodiments, the polymerase can be unlabeled. Alternatively, thepolymerase can be linked to one or more label to form a labeledpolymerase conjugate. In some embodiments, the label comprises at leastone energy transfer moiety. The label can be an organic label (e.g., dyelabel) or an inorganic label (e.g., nanoparticle).

In some embodiments, the polymerase may be linked with at least oneenergy transfer donor moiety. One or more energy transfer donor moietiescan be linked to the polymerase at the amino end or carboxyl end or maybe inserted at any site therebetween. Optionally, the energy transferdonor moiety can be attached to the polymerase in a manner which doesnot significantly interfere with the nucleotide binding activity, orwith the nucleotide incorporation activity of the polymerase. In suchembodiments, the energy transfer moiety is attached to the polymerase ina manner that does not significantly interfere with polymerase activity.

In one embodiment, a single energy transfer donor moiety can be linkedto more than one polymerase and the attachment can be at the amino endor carboxyl end or may be inserted within the polymerase.

In another embodiment, a single energy transfer donor moiety can belinked to one polymerase.

In one embodiment, the energy transfer donor moiety can be ananoparticle (e.g., a fluorescent nanoparticle) or a fluorescent dye.The polymerase, which can be linked to the nanoparticle or fluorescentdye, typically retains one or more activities that are characteristic ofthe polymerase, e.g., polymerase activity, exonuclease activity,nucleotide binding, and the like.

In some embodiments, the polymerases of the present disclosure caninclude one or more mutations that improve the performance of thepolymerase in the particular biological assay of interest. The mutationscan include amino acid substitutions, insertions, or deletions.

Selecting a Polymerase

The selection of the polymerase for use in the disclosed methods can bebased on the desired polymerase behavior in the particular biologicalassay of interest. For example, the polymerase can be selected toexhibit enhanced or reduced activity in a particular assay, or enhancedor reduced interaction with one or more particular substrates.

For example, in some embodiments the polymerase can be selected based onthe polymerization kinetics of the polymerase either in unconjugatedform or when linked to a label (labeled polymerase conjugate).Optionally, the label can be a nanoparticle or fluorescent dye; in someembodiments, the label can be energy transfer donor moiety. For example,the polymerase can be selected on the basis of kinetic behavior relatingto nucleotide binding (e.g., association), nucleotide dissociation(intact nucleotide), nucleotide fidelity, nucleotide incorporation(e.g., catalysis), and/or release of the cleavage product. The selectedpolymerase can be wild-type or mutant.

In one embodiment, polymerases may be selected that retain the abilityto selectively bind complementary nucleotides. In another embodiment,the polymerases may be selected which exhibit a modulated rate (fasteror slower) of nucleotide association or dissociation. In anotherembodiment, the polymerases may be selected which exhibit a reduced rateof nucleotide incorporation activity (e.g., catalysis) and/or a reducedrate of dissociation of the cleavage product and/or a reduced rate ofpolymerase translocation (after nucleotide incorporation). Some modifiedpolymerases which exhibit modified nucleotide binding and/or rates ofnucleotide incorporation, as well as methods of identifying suchpolymerases, have been described. See, e.g., Rank, U.S. Published PatentApplication No. 2008/0108082; Hanzel, U.S. Published Patent ApplicationNo. 2007/0196846; Clark, U.S. Published Patent Application No.2009/0176233; Bjornsen, U.S. Published Patent Application No.2009/0286245.

In polymerases from different classes (including DNA-dependentpolymerases), an active-site lysine can interact with the phosphategroups of a nucleoside triphosphate molecule bound to the active site.The lysine residue has been shown to protonate the pyrophosphateleaving-group upon nucleotidyl transfer. Mutant polymerases having thislysine substituted with leucine, arginine, histidine or other aminoacids, exhibit greatly reduced nucleotide incorporation rates (Castro,et al., 2009 Nature Structural and Molecular Biology 16:212-218). Oneskilled in the art can use amino acid alignment and/or comparison ofcrystal structures of polymerases as a guide to determine which lysineresidue to replace with alternative amino acids. The sequences of Phi-29polymerase (SEQ ID NO: 1), RB69 polymerase (SEQ ID NO: 15), an exemplaryB103-like polymerase (SEQ ID NO: 7), and Klenow fragment can be used asthe basis for selecting the amino acid residues to be modified (for B103polymerase, see Hendricks, et al., U.S. Ser. No. 61/242,771, filed onSep. 15, 2009, or U.S. Ser. No. 61/293,618, filed on Jan. 8, 2010). Inone embodiment, a modified Phi-29 polymerase can include lysine atposition 379 and/or 383 substituted with leucine, arginine or histidine.

In other embodiments, the polymerase can be selected based on thecombination of the polymerase and nucleotides, and the reactionconditions, to be used for the nucleotide binding and/or nucleotideincorporation reactions. For example, certain polymerases in combinationwith nucleotides that comprise 3, 4, 5, 6, 7, 8, 9, 10 or more phosphategroups can be selected for performing the disclosed methods. In anotherexample, certain polymerases in combination with nucleotides which arelinked to an energy transfer moiety can be selected for performing thenucleotide incorporation methods.

In some embodiments, the modified polymerase can be attached to orassociated with a substrate or surface. In some embodiments, thepolymerase can be attached to or associated with a nucleic acid molecule(termed a template), and polymerize one or more nucleotides in atemplate-dependent fashion. In some embodiments, the template can beattached to or associated with a substrate or surface. In someembodiments, the polymerase, template, nucleotide, substrate or surface,or some combination thereof, can also be labeled.

In some embodiments, the methods of the present invention can beperformed in multiplex and/or “high-throughput” format wherein multipleunits of the modified polymerases of the present disclosure can each bevisualized and monitored in parallel with each other. For example, insome embodiments, multiple modified polymerases may be positioned,associated with, or attached to different locations on a substrate, anda polymerase activity of one or more of these polymerases may bedetected in isolation. In some embodiments, the polymerase or thetemplate nucleic acid molecule are associated with or attached to asubstrate or surface in array format. The array can be spatiallyaddressable.

In some embodiments, the nucleic acid molecule, the, modifiedpolymerase, or both, may be isolated within a suitable nanostructure.i.e., a structure having at least one dimension measuring 500 nm orless. The nanostructure can be useful in isolating a single nucleic acidmolecule or polymerase. In some embodiments, the nanostructure can beuseful in elongating the nucleic acid molecule to permit visualizationof nucleotide synthesis along some or all of the length of the nucleicacid molecule. In some embodiments, the nanostructure is also useful inlimiting the amount of background signal (“noise”) in the system byreducing the excitation or detection volume, and/or by reducing theamount of labeled moieties present within the reaction chamber. In someembodiments, the nanostructure is designed to admit only a singlepolymeric molecule and elongate it as it flows through thenanostructure. Suitable devices comprising nanostructures that may beused to practice the inventions disclosed herein are described, forexample, in U.S. Pat. Nos. 6,635,163; 7,217,562, U.S. Pub. No.2004/0197843 and U.S. Pub. No. 2007/0020772. In some embodiments, thenanostructures of the nanofluidic device will satisfy threerequirements: (1) they will have a sufficiently small dimension toelongate and isolate macromolecules; (2) they will be sufficient lengthto permit instantaneous observation of the entire elongatedmacromolecule; and (3) the nanochannels or other nanostructures will besufficiently numerous to permit simultaneous and parallel observation ofa large population of macromolecules. In one embodiment, the radius ofthe component nanostructures of the nanofluidic device will be roughlyequal to or less than the persistence length of the target DNA. Suitablemethods of detecting nucleotide incorporations using nanostructures aredisclosed, for example, in U.S. Provisional Application Nos. 61/077,090,filed Jun. 30, 2008; 61/089,497, filed Aug. 15, 2008; and 61/090,346,filed Aug. 20, 2008; and International Application No. PCT/US09/049324,filed Jun. 30, 2009.

Typically, the polymerase reaction comprises a mixture including amodified polymerase, a nucleic acid molecule, at least one priming sitefor nucleotide polymerization, and one or more nucleotides for themodified polymerase. In some embodiments, the reaction can be initiatedby preparing the mixture lacking one essential component forpolymerization (for example, the polymerase or nucleotides) and thenadding the withheld component to initiate the reaction. Suitabletemperatures and the addition of other components such as divalent metalions can be determined and optimized based on the particular polymeraseand the target nucleic acid sequences.

In some embodiments, the polymerase reaction can be performed usingbuffer conditions comprising 50 mM Tris buffer pH 7.5, 50 mM NaCl, 10 mMMgCl₂, 0.5 mM MnCl₂. In some embodiments, 0.3% BSA and/or 0.05% Tween20can be included in the reaction mix. In some embodiments, the reactionmix is further supplemented with 2 mM DTT and/or single stranded bindingprotein (SSBP) at a concentration of 100 μg/ml. Alternatively, thepolymerase reaction can be performed using buffer conditions comprising50 mM Tris pH 8.0, 50 mM NaCl and 10 mM MgCl₂. In some embodiments,divalent cations, such as calcium, can be added to the polymerasereaction in an amount sufficient to block further nucleotideincorporation. See, e.g., U.S. Provisional Application 61/242,762, filedSep. 15, 2009.

In some embodiments, a suitable primer is included in the nucleic acidpolymerase reaction. The primer length is typically determined by thespecificity desired for binding the complementary template as well asthe stringency of the annealing and reannealing conditions employed. Theprimer can be synthetic, or produced naturally by primases, RNApolymerases, or other oligonucleotide synthesizing enzymes. The primercan be any suitable length including at least 5 nucleotides, 5 to 10,15, 20, 25, 50, 75, 100 nucleotides or longer in length. In someembodiments, the polymerase extends the primer by a plurality ofnucleotides. In some embodiments, the primer is extended at least 50,100, 250, 500, 1000, or at least 2000 nucleotide monomers.Alternatively, the initiation site for nucleotide polymerization can becreated through any suitable means without requiring use of a primer.For example, the polymer to be sequenced can comprise, or be associatedwith, a polymerase priming site capable of extension via polymerizationof monomers by the polymerase. The priming site can be generated, forexample, by treatment of the polymer so as to produce nicks or cleavagesites. Yet another option is for the target polymer to undergo “hairpin”formation, either through annealing to a self-complementary regionwithin the target sequence itself or through ligation to aself-complementary sequence, resulting in a structure that undergoesself-priming under suitable conditions. Alternatively, the priming canbe facilitated through the use of various accessory proteins known tofacilitate priming of DNA synthesis by a given polymerase, such as theterminal protein of Phi-29 DNA polymerase and/or B103 DNA polymerase.See, e.g., M. Salas, “Protein-priming of DNA replication”, Ann. Rev.Biochem. 60:39-71 (1991).

In some embodiments, the labeled nucleotide is a nucleotide analogcomprising a label linked to the base, sugar, phosphate or any otherportion of the nucleotide analog. In some embodiments, the nucleotidecan also comprise a blocking group that inhibits, slows down or blocksfurther incorporation of nucleotides onto the end of the nucleic acidmolecule until the blocking group is removed from the nucleotide. Insome embodiments, the blocking group can be removed from the nucleotideby exposure to chemical, enzymatic, or photocleaving agents. In someembodiments, the method further includes the step of adding one or moredivalent cations to the polymerase reaction mixture in an amountsufficient for inhibiting further incorporation of nucleotides onto theend of the nucleic acid molecule by the modified polymerase. Suchmethods are described, for example, in U.S. Provisional Application No.61/242,762, filed Sep. 15, 2009; and U.S. Provisional Application No.61/184,774, filed on Jun. 5, 2009.

In some embodiments, the methods, compositions, systems and/or kitsdisclosed herein can involve the use of one or more moieties capable ofundergoing energy transfer, for example resonance energy transfer (RET).Such energy transfer moieties can include resonance energy transferdonors and acceptors. The energy transfer moieties can be linked to thesolid surfaces, nanoparticles, polymerases, nucleotides, target nucleicacid molecules, primers, and/or oligonucleotides.

In one aspect, the energy transfer moiety can be an energy transferdonor. For example, the energy transfer donor can be a nanoparticle oran energy transfer donor moiety (e.g., fluorescent dye). In anotheraspect, the energy transfer moiety can be an energy transfer acceptor.For example, the energy transfer acceptor can be an energy acceptor dye.In another aspect, the energy transfer moiety can be a quencher moiety.

In one aspect, the energy transfer pair can be linked to the samemolecule. For example, the energy transfer donor and acceptor pair canbe linked to a single polymerase, which can provide detection ofconformational changes in the polymerase. In another aspect, the donorand acceptor can be linked to different molecules in any combination.For example, the donor can be linked to the polymerase, target molecule,or primer molecule, and/or the acceptor can be linked to the nucleotide,the target molecule, or the primer molecule.

The energy transfer donor is capable of absorbing electromagnetic energy(e.g., light) at a first wavelength and emitting excitation energy inresponse. The energy acceptor is capable of absorbing excitation energyemitted by the donor and fluorescing at a second wavelength in response.

The donor and acceptor moieties can interact with each other physicallyor optically in a manner which produces a detectable signal when the twomoieties are in proximity with each other. A proximity event includestwo different moieties (e.g., energy transfer donor and acceptor)approaching each other, or associating with each other, or binding eachother.

The donor and acceptor moieties can transfer energy in various modes,including: fluorescence resonance energy transfer (FRET) (L. Stryer 1978Ann. Rev. Biochem. 47: 819-846; Schneider, U.S. Pat. No. 6,982,146;Hardin, U.S. Pat. No. 7,329,492; Hanzel U.S. published patentapplication No. 2007/0196846), scintillation proximity assays (SPA)(Hart and Greenwald 1979 Molecular Immunology 16:265-267; U.S. Pat. No.4,658,649), luminescence resonance energy transfer (LRET) (G. Mathis1995 Clin. Chem. 41:13914397), direct quenching (Tyagi et al, 1998Nature Biotechnology 16:49-53), chemiluminescence energy transfer (CRET)(Campbell and Patel 1983 Biochem. Journal 216:185494), bioluminescenceresonance energy transfer (BRET) (Y. Xu, et al., 1999 Proc. Natl. Acad.Sci. 96:151456), and excimer formation (J. R. Lakowicz 1999 “Principlesof Fluorescence Spectroscopy”, Kluwer Academic/Plenum Press, New York).

In one exemplary embodiment, the energy transfer moieties can be a FRETdonor/acceptor pair. FRET is a distance-dependent radiationlesstransmission of excitation energy from a first moiety, referred to as adonor moiety, to a second moiety, referred to as an acceptor moiety.Typically, the efficiency of FRET energy transmission is dependent onthe inverse sixth-power of the separation distance between the donor andacceptor, r. For a typical donor-acceptor pair, r can vary betweenapproximately 10400 Angstroms. FRET is useful for investigating changesin proximity between and/or within biological molecules. In someembodiments, FRET efficiency may depend on donor-acceptor distance r as1/r⁶ or 1/r⁴. The efficiency of FRET energy transfer can sometimes bedependent on energy transfer from a point to a plane which varies by thefourth power of distance separation (E. Jares-Erijman, et al., 2003 Nat.Biotechnol. 21:1387). The distance where FRET efficiency is at 50% istermed R₀, also know as the Forster distance. R₀ can be unique for eachdonor-acceptor combination and can range from between about 5 nm toabout 10 nm. A change in fluorescence from a donor or acceptor during aFRET event (e.g., increase or decrease in the signal) can be anindication of proximity between the donor and acceptor.

FRET efficiency (E) can be defined as the quantum yield of the energytransfer transition, i.e. the fraction of energy transfer eventoccurring per donor excitation event. It is a direct measure of thefraction of photon energy absorbed by the donor which is transferred toan acceptor, as expressed in Equation 1: E=k_(ET)/k_(f)+k_(ET)+Σk_(i)where k_(ET) is the rate of energy transfer, k_(f) the radiative decayrate and the k_(i) are the rate constants of any other de-excitationpathway.

FRET efficiency E generally depends on the inverse of the sixth power ofthe distance r (nm) between the two fluorophores (i.e., donor andacceptor pair), as expressed in Equation 2: E=1/1+(r/R₀)⁶.

Therefore, the FRET efficiency of a donor describes the maximumtheoretical fraction of photon energy which is absorbed by the donor(i.e., nanoparticle) and which can then be transferred to a typicalorganic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).

In biological applications, FRET can provide an on-off type signalindicating when the donor and acceptor moieties are proximal (e.g.,within R₀) of each other. Additional factors affecting FRET efficiencyinclude the quantum yield of the donor, the extinction coefficient ofthe acceptor, and the degree of spectral overlap between the donor andacceptor. Procedures are well known for maximizing the FRET signal anddetection by selecting high yielding donors and high absorbing acceptorswith the greatest possible spectral overlap between the two (D. W.Piston and G. J. Kremers 2007 Trends Biochem. Sci. 32:407). Resonanceenergy transfer may be either an intermolecular or intramolecular event.Thus, the spectral properties of the energy transfer pair as a whole,change in some measurable way if the distance and/or orientation betweenthe moieties are altered.

The production of signals from FRET donors and acceptors can besensitive to the distance between donor and acceptor moieties, theorientation of the donor and acceptor moieties, and/or a change in theenvironment of one of the moieties (Deuschle et al. 2005 Protein Science14: 2304-2314; Smith et al. 2005 Protein Science 14:64-73). For example,a nucleotide linked with a FRET moiety (e.g., acceptor) may produce adetectable signal when it approaches, associates with, or binds apolymerase linked to a FRET moiety (e.g., donor). In another example, aFRET donor and acceptor linked to one protein can emit a FRET signalupon conformational change of the protein. Some FRET donor/acceptorpairs exhibit changes in absorbance or emission in response to changesin their environment, such as changes in pH, ionic strength, ionic type(NO₂. Ca⁺², Mg⁺², Zn⁺², Na⁺, Cl⁻, K⁺), oxygen saturation, and solvationpolarity.

The FRET donor and/or acceptor may be a fluorophore, luminophore,chemiluminophore, bioluminophore, or quencher (P. Selvin 1995 MethodsEnzymol 246:300-334; C. G. dos Remedios 1995 J. Struct. Biol.115:175-185; P. Wu and L. Brand 1994 Anal Biochem 218:1-13).

In some embodiments, the energy transfer moieties may not undergo FRET,but may undergo other types of energy transfer with each other,including luminescence resonance energy transfer, bioluminescenceresonance energy transfer, chemiluminescence resonance energy transfer,and similar types of energy transfer not strictly following theForster's theory, such as the non-overlapping energy transfer whennon-overlapping acceptors are utilized (Laitala and Hemmila 2005 Anal.Chem. 77: 1483-1487).

In one embodiment, the modified polymerases as provided herein can belinked to an energy transfer moiety, for example an energy transferdonor moiety. In another embodiment, the nucleotide can be linked to anenergy transfer acceptor moiety. For example, in one embodiment thenucleotide comprises a polyphosphate chain and an energy transfer moietylinked to the terminal phosphate group of the polyphosphate chain. Achange in a fluorescent signal can occur when the labeled nucleotide isproximal to the labeled polymerase.

In one embodiment, when an acceptor-labeled nucleotide is proximal to adonor-labeled modified polymerase as provided herein, the signal emittedby the donor moiety decreases. In another embodiment, when theacceptor-labeled nucleotide is proximal to the donor-labeled polymerase,the signal emitted by the acceptor moiety increases. In anotherembodiment, a decrease in donor signal and increase in acceptor signalcorrelates with nucleotide binding to the polymerase and/or correlateswith polymerase-dependent nucleotide incorporation.

Quenchers

In some embodiments, the energy transfer moiety can be a FRET quencher.Typically, quenchers have an absorption spectrum with large extinctioncoefficients, however the quantum yield for quenchers is reduced, suchthat the quencher emits little to no light upon excitation. Quenchingcan be used to reduce the background fluorescence, thereby enhancing thesignal-to-noise ratio. In one aspect, energy transferred from the donormay be absorbed by the quencher which emits moderated (e.g., reduced)fluorescence. In another aspect, the acceptor can be a non-fluorescentchromophore which absorbs the energy transferred from the donor andemits heat (e.g., the energy acceptor is a dark quencher).

For an example, a quencher can be used as an energy acceptor with ananoparticle donor in a FRET system, see I. L. Medintz, et al., 2003Nature Materials 2:630. One exemplary method of primer extension usingthe modified polymerases of the present disclosure involves the use ofquenchers in conjunction with reporters comprising fluorescent reportermoieties. In this strategy, certain nucleotides in the reaction mixtureare labeled with a reporter comprising a fluorescent label, while theremaining nucleotides are labeled with one or more quenchers.Alternatively, each of the nucleotides in the reaction mixture islabeled with one or more quenchers. Discrimination of the nucleotidebases is based on the wavelength and/or intensity of light emitted fromthe FRET acceptor, as well as the intensity of light emitted from theFRET donor. If no signal is detected from the FRET acceptor, acorresponding reduction in light emission from the FRET donor indicatesincorporation of a nucleotide labeled with a quencher. The degree ofintensity reduction may be used to distinguish between differentquenchers.

Examples of fluorescent donors and non-fluorescent acceptor (e.g.,quencher) combinations have been developed for detection of proteolysis(Matayoshi 1990 Science 247:954-958) and nucleic acid hybridization (L.Morrison, in: Nonisotopic DNA Probe Techniques, ed., L. Kricka, AcademicPress, San Diego, (1992) pp. 311-352; S. Tyagi 1998 Nat. Biotechnol.16:49-53; S. Tyagi 1996 Nat. Biotechnol. 14:947-8). FRET donors,acceptors and quenchers can be moieties which absorb electromagneticenergy (e.g., light) at about 300-900 nm, or about 350-800 nm, or about390-800 nm.

Materials for Energy Transfer Moieties

Energy transfer donor and acceptor moieties can be made from materialswhich typically fall into four general categories (see the review in: K.E. Sapford, et al., 2006 Angew. Chem. Int. Ed. 45:4562-4588); including:(1) organic fluorescent dyes, dark quenchers and polymers (e.g.,dendrimers); (2) inorganic material such as metals, metal chelates andsemiconductors nanoparticles; (3) biomolecules such as proteins andamino acids (e.g., green fluorescent protein and derivatives thereof);and (4) enzymatically catalyzed bioluminescent molecules. The materialfor making the energy transfer donor and acceptor moieties can beselected from the same or different categories.

The FRET donor and acceptor moieties which are organic fluorescent dyes,quenchers or polymers can include traditional dyes which emit m the UV,visible, or near-infrared region. The UV emitting dyes includecoumarin-, pyrene-, and naphthalene-related compounds. The visible andnear-infrared dyes include xanthene-, fluorescein-, rhodol-, rhodamine-,and cyanine-related compounds. The fluorescent dyes also includes DDAO((7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)), resorufin,ALEXA FLUOR and BODIPY dyes (both Molecular Probes), HILYTE Fluors(AnaSpec), ATTO dyes (Alto-Tec), DY dyes (Dyonics GmbH); TAMRA (PerkinElmer), tetramethylrhodamine (TMR), TEXAS RED, DYLIGHT (Thermo FisherScientific), FAM (AnaSpec), JOE and ROX (both Applied Biosystems), andTokyo Green.

Additional fluorescent dyes which can be used as quenchers includes:DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ (BiosearchTechnologies), QXL (AnaSpec), BBQ (Berry and Associates) and CY5Q/7Q(Amersham Biosciences).

The FRET donor and acceptor moieties which comprise inorganic materialsinclude gold. (e.g., quencher), silver, copper, silicon, semiconductornanoparticles, and fluorescence-emitting metal such as a lanthanidecomplex, including those of Europium and Terbium.

Suitable FRET donor/acceptor pairs include: FAM as the donor and JOE,TAMRA, and ROX as the acceptor dyes. Other suitable pairs include: CYAas the donor and R6G, TAMRA, and ROX as the donor dyes. Other suitabledonor/acceptor pairs include: a nanoparticle as the donor, and ALEXAFLUORS dyes (e.g., 610, 647, 660, 680, 700). DYOMICS dyes, such as 634and 734 can be used as energy transfer acceptor dyes.

In some embodiments, the energy transfer moieties undergo RET, (forexample, FRET) that is characterized by the generation of a signal,herein termed a RET or FRET signal, as the case may be. The RET or FRETsignal can be an optically detectable signal, for example, an increasein acceptor fluorescence or a decrease in donor fluorescence.

In some embodiments, the modified polymerase as provided herein islinked to a FRET donor and contacted with one or more labeled nucleotideanalogs, wherein the label of the one or more labeled nucleotidescomprises a FRET acceptor. In some embodiments, interaction of thelabeled nucleotide with the labeled polymerase (occurring, for example,in association with a productive incorporation, a non-productiveincorporation or during association of a nucleotide with the polymeraseactive site) results in the emission of a FRET signal. The FRET signalcan optionally be detected and analyzed to determine the occurrence of apolymerase-nucleotide interaction.

Typically, the one or more signals indicative of polymerase-nucleotideinteraction are one or more FRET signals resulting from FRET between thelabel of the modified polymerase and the label of an incorporatingnucleotide. The FRET can occur prior to, during or after productiveincorporation of the nucleotide into a nucleic acid molecule.Alternatively, the FRET can occur prior to binding of the nucleotide tothe polymerase active site, or while the nucleotide resides within thepolymerase active site, during a non-productive incorporation.

In some embodiments, the modified polymerase comprises a FRET donormoiety and one or more nucleotides comprises a FRET acceptor moiety. TheFRET acceptor moiety can in some embodiments be attached to, or comprisepart of, the nucleotide sugar, the nucleobase, or analogs thereof.

In some embodiments, at least one nucleotide of the polymerase reactionis labeled with a nucleotide label. In some embodiments, the nucleotidelabel is linked to the nucleotide using a linker and/or spacer usingsuitable techniques. Any suitable methods for labeling nucleotides canbe employed including but not limited to those described in U.S. Pat.Nos. 7,041,892, 7,052,839, 7,125,671 and 7,223,541; U.S. Pub. Nos.2007/0072196 and 2008/0091005; Sood et al., 2005, J. Am. Chem. Soc.127:2394-2395; Arzumanov et al., 1996, J. Biol. Chem. 271:24389-24394;and Kumar et al., 2005, Nucleosides, Nucleotides & Nucleic Acids,24(5):401-408. Suitable labels that can be used in the disclosed methodsand linked to the polymerase or the nucleotides include any molecule,nano-structure, or other chemical structure that capable of beingdetected by a detection system, including but not limited to fluorescentdyes.

In some embodiments, the nucleotide label is attached to a portion ofthe nucleotide that is released upon incorporation of the underlyingnucleotide. For example, in some embodiments, the nucleotide label isattached to a portion of the nucleotide that is released uponincorporation of the underlying nucleotide, For example, in someembodiments, the nucleotide comprises a polyphosphate chain and the FRETacceptor is attached to a phosphate group of the nucleotide that iscleaved and released upon incorporation of the underlying nucleotideinto the primer strand, for example the γ-phosphate, the β-phosphate orsome other terminal phosphate of the incoming nucleotide. When thisacceptor-labeled nucleotide polyphosphate is incorporated by themodified polymerase into a nucleic acid molecule, the polymerase cleavesthe bond between the alpha and beta phosphate, thereby releasing apyrophosphate moiety comprising the acceptor that diffuses away. Thus,in these embodiments, a signal indicative of nucleotide incorporation isgenerated through FRET between the nanoparticle and the acceptor bondedto the gamma, beta or other terminal phosphate as each incomingnucleotide is incorporated into the newly synthesized strand. Bycleavage of the terminal phosphate(s) and release of the label uponincorporation of the incoming nucleotide, the FRET signal from the labelceases after the nucleotide is incorporated and the label diffuses away.By releasing the label upon incorporation, successive incorporation oflabeled nucleotides can each be detected without interference fromnucleotides previously incorporated into the complementary strand.

Alternatively, the nucleotide label can be linked to the alpha (a)phosphate, the beta 03) phosphate, another internal phosphate, base,sugar or any other portion of the nucleotide that typically becomesincorporated into the growing nucleic acid molecule. Although suchlabels typically are typically not cleaved and released during theincorporation process, and thus become incorporated into the growingnucleic acid molecule, they can optionally be removed and/or renderedinoperable via suitable treatments, e.g., chemical cleavage, enzymaticcleavage and/or photobleaching, later in the process. In someembodiments, the portion of the nucleotide that remains in the extendingnucleic acid molecule after the label and/or blocking group is releasedor otherwise removed is structurally similar or identical to the portionincorporated from a natural nucleotide; alternatively, the incorporatedportion may contain structural or chemical elements that are differentfrom the incorporated portion of a natural nucleotide.

In some embodiments, a signal indicative of nucleotide incorporation isgenerated as each incoming nucleotide becomes incorporated by thepolymerase of the conjugate. In embodiments where the nucleotide labelis cleaved and released upon nucleotide incorporation, successiveextensions can each be detected without interference from nucleotidespreviously incorporated into the complementary strand.

In some embodiments, the nucleotide and nucleotide label are linkedusing a linker. The linker can include multiple amino acid residues(e.g., arginine) that serve as an intervening linker between thenucleotide and the nucleotide label. For example, the linker cancomprise four arginine residues that connect a dye label to a terminalphosphate group of the nucleotide.

In other embodiments, the label linked or attached to the nucleotide canbe a quencher. Quenchers are useful as acceptors in FRET applications,because they produce a signal through the reduction or quenching offluorescence from the donor fluorophore. For example, in aquencher-based system, illumination of the donor fluorophore excites thedonor, and if an appropriate acceptor is not close enough to the donor,the donor fluoresces. This fluorescence is reduced or abolished when aquencher is in sufficient proximity to quench the donor, therebyreducing or abolishing donor fluorescence. Thus, interaction orproximity between a donor and quencher-acceptor can be detected by thereduction or absence of donor fluorescence. For an example of the use ofa quencher as an acceptor with a nanoparticle donor, see, e.g., Medintz,I. L. et al. (2003) Nat. Mater. 2:630. Examples of quenchers include theQSY dyes available from Molecular Probes (Eugene, Oreg.).

One exemplary method of primer extension using the modified polymerasesof the present disclosure involves the use of quenchers in conjunctionwith fluorescent labels. In some embodiments, certain nucleotides in thereaction mixture are labeled with a fluorescent label, while theremaining nucleotides are labeled with one or more quenchers.Alternatively, each of the nucleotides in the reaction mixture islabeled with one or more quenchers. Discrimination of the nucleotidebases is based on the wavelength and/or intensity of light emitted fromthe FRET acceptor, as well as the intensity of light emitted from theFRET donor. If no signal is detected from the FRET acceptor, acorresponding reduction in light emission from the FRET donor indicatesincorporation of a nucleotide labeled with a quencher. The degree ofintensity reduction can be used to distinguish between differentquenchers.

Typically, the label of the modified polymerase and/or the label of thenucleotide will be selected and/or designed to minimize any adverseeffect of such labels on the progress of the polymerization reaction asdetermined by speed, error rate, fidelity, processivity and average readlength of the newly synthesized strand.

In some embodiments, the nucleotide can comprise a moiety that altersthe functional properties of the nucleotide. For example, the moiety caninterfere or impede the ability of the nucleotide to enter thepolymerase active site, become released from the polymerase active sitefollowing incorporation of the nucleotide onto the end of an extendingnucleic acid molecule, or become covalently linked to the end of theextending nucleic acid molecule. In some embodiments, the moiety canaffect the kinetic properties of the nucleotide, such as, for example,modify the K_(m), V_(max), residence time or branching ratio of thenucleotide with one or more polymerases of the present disclosure.

In some embodiments, the nucleotide is a labeled analog of a naturallyoccurring nucleotide. The label can be an optically detectable label.For example, the label can be a photoluminescent, chemiluminescent,bioluminescent, fluorescent or fluorogenic moiety. The label can be amass tag or molecular volume tag. In some embodiments, the label is aFRET moiety. In some embodiments, the identity of the label correlateswith the base identity of the nucleotide. In some embodiments, the labelis capable of emitting a signal that correlates with the base identityof the nucleotide. In some embodiments, the label of the labelednucleotide is a chromophore, fluorophore or luminophore. In someembodiments, the label of the labeled nucleotide can be a fluorophore orfluorogen selected from the group consisting of: xanthine dye,fluorescein, cyanine, rhodamine, coumarin, acridine, Texas Red dye,BODIPY, ALEXA (Molecular Probes/Invitrogen, Life Technologies Corp.),GFP, and a derivative or modification of any of the foregoing. Someexamples of suitable labels are described, for example, in International(PCT) Published Application No. WO/2008/030115; Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog(Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2ndEd., Plenum Press New York (1999), all of which are incorporated hereinby reference in their entireties.

In some embodiments, incorporation of the nucleotide into an extendingnucleic acid molecule by a polymerase can generate a nucleic acidmolecule comprising non-standard moieties, i.e., moieties that are nottypically present in naturally occurring nucleic acid molecules. Forexample, the synthesized nucleic acid molecule may comprise one or morelabels and/or include one or more atoms, for example sulfur or boronatoms, not typically present in naturally occurring nucleic acidmolecules. In some embodiments, the label is linked to a base, sugar orphosphate moiety of the nucleotide via a linker.

The primer extension and/or single molecule sequencing methods using themodified polymerases of the present disclosure can be practiced usingnucleotides. In some embodiments, the nucleotide can comprise a moietythat facilitates purification or detectability of the nucleotide. Insome embodiments, the moiety is a label. The label can in someembodiments be linked to a base, sugar or phosphate moiety. In someembodiments, the nucleotide is a nucleotide polyphosphate and the labelis linked to the terminal phosphate of the nucleotide polyphosphate.

In some embodiments the nucleotides can be linked with at least oneenergy transfer moiety. The energy transfer moiety can be an energytransfer acceptor moiety. The different types of nucleotides (e.g.,adenosine, thymidine, cytidine, guanosine, and uridine) can be labeledwith different energy transfer acceptor moieties so that the detectablesignals from each of the different types of nucleotides can bedistinguishable to permit base identity. The nucleotides can be labeledin a way that does not interfere with the events of polymerization. Forexample the attached energy transfer acceptor moiety does not interferewith nucleotide binding and/or does not interfere with nucleotideincorporation and/or does not interfere with cleavage of thephosphodiester bonds and/or does not interfere with release of thepolyphosphate product. See for example, U.S. Ser. No. 61/164,091, RonaldGraham, filed Mar. 27, 2009. See for example U.S. Pat. Nos. 7,041,812,7,052,839, 7,125,671, and 7,223,541; U.S. Pub. Nos. 2007/0072196 and2008/0091005; Sood et al., 2005, J. Am. Chem. Soc. 127:2394-2395;Arzumanov et al., 1996, J. Biol. Chem. 271:24389-24394; and Kumar etal., 2005, Nucleosides, Nucleotides & Nucleic Acids, 24(5):401-408.

In one aspect, the energy transfer acceptor moiety may be linked to anyposition of the nucleotide.

For example, the energy transfer acceptor moiety can be linked to anyphosphate group (or derivatized phosphate group), the sugar or the base.In another example, the energy transfer moiety can be linked to anyphosphate group (or derivatized phosphate group) which is released aspart of a phosphate cleavage product upon incorporation. In yet anotherexample, the energy transfer acceptor moiety can be linked to theterminal phosphate group (or derivatized phosphate group). In anotheraspect, the nucleotide may be linked with an additional energy transferacceptor moiety, so that the nucleotide is attached with two or moreenergy transfer acceptor moieties. The additional energy transferacceptor moiety can be the same or different as the first energytransfer acceptor moiety. In one embodiment, the energy transferacceptor moiety can be a FRET acceptor moiety.

In one aspect, the nucleotide may be linked with a reporter moiety whichis not an energy transfer moiety. For example, the reporter moiety canbe a fluorophore.

In one aspect, the energy transfer acceptor moieties and/or the reportermoiety can be attached to the nucleotide via a linear or branched linkermoiety. An intervening linker moiety can connect the energy transferacceptor moieties with each other and/or to the reporter moiety, anycombination of linking arrangements.

In another aspect, the nucleotides comprise a sugar moiety, base moiety,and at least three, four, five, six, seven, eight, nine, ten, or morephosphate groups linked to the sugar moiety by an ester or phosphoramidelinkage. The phosphates can be linked to the 3′ or 5′ C of the sugarmoiety. The nucleotides can be incorporated and/or polymerized into agrowing nucleic acid strand by a naturally occurring, modified, orengineered nucleic acid dependent polymerase.

In one aspect, different linkers can be used to operably link thedifferent nucleotides (e.g., A, G, C, or T/U) to the energy transfermoieties or reporter moieties. For example, adenosine nucleotide can beattached to one type of energy transfer moiety using one type of linker,and guanosine nucleotide can be linked to a different type of energytransfer moiety using a different type of linker. In another example,adenosine nucleotide can be attached to one type of energy transfermoiety using one type of linker, and the other types of nucleotides canbe attached to different types of energy transfer moieties using thesame type of linker. One skilled in the art will appreciate that manydifferent combinations of nucleotides, energy transfer moieties, andlinkers are possible.

In one aspect, the distance between the nucleotide and the energytransfer moiety can be altered. For example, the linker length and/ornumber of phosphate groups can lengthen or shorten the distance from thesugar moiety to the energy transfer moiety. In another example, thedistance between the nucleotide and the energy transfer moiety candiffer for each type of nucleotide (e.g., A, G, C, or T/U).

In another aspect, the number of energy transfer moieties which arelinked to the different types of nucleotides (e.g., A, G, C, or T/U) canbe the same or different. For example: A can have one dye, and G, C, andT have two; A can have one dye, C has two, G has three, and T has four;A can have one dye, C and G have two, and T has four. One skilled in theart will recognize that many different combinations are possible.

In another aspect, the concentration of the labeled nucleotides used toconduct the nucleotide binding or nucleotide incorporation reactions canbe about 0.0001 nM-1 μM, or about 0.0001 nM-0.001 nM, or about 0.001nM-0.01 nM, or about 0.01 nM-0.1 nM, or about 0.1 nM-1.0 nM, or about 1nM-25 nM, or about 25 nM-50 nM, or about 50 nM-75 nM, or about 75 nM-100nM, or about 100 nM-200 nM, or about 200 nM-500 nM, or about 500 nM-750nM, or about 750 nM-1000 nM, or about 0.1 μM-20 μM, or about 20 μM-50μM, or about 50 μM-75 μM, or about 75 μM-100 μM, or about 100 μM-200 μM,or about 200 μM-500 μM, or about 500 μM-750 μM, or about 750 μM-1000 μM.

In another aspect, the concentration of the different types of labelednucleotides, which are used to conduct the nucleotide binding orincorporation reaction, can be the same or different from each other.

Sugar Moieties

The nucleotides typically comprise suitable sugar moieties, such ascarbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48),acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal ChemistryLetters vol. 7: 3013-3016), and other suitable sugar moieties (Joeng, etal., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem.36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No.5,558,991). The sugar moiety may be selected from the following:ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl,2′,3′-didehydrodideoxyribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl,2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoriboxyl,2′-alkylthioribosyl, 3′-alkoxyribosyl, 3′-azidoribosyl, 3′-aminoribosyl,3′-fluororibosyl, 3′-mercaptoriboxyl, 3′-alkylthioribosyl carbocyclic,acyclic and other modified sugars. In one aspect, the 3′-position has ahydroxyl group, for strand/chain elongation.

Base Moieties

The nucleotides can include a hetero cyclic base which includessubstituted or unsubstituted nitrogen-containing parent heteroaromaticring which is commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants. Thebase is capable of forming Watson-Crick and/or Hoogstein hydrogen bondswith an appropriate complementary base. Exemplary bases include, but arenot limited to, purines and pyrimidines such as: 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-Δ²-isopentenyladenine(6 iA), N⁶-Δ²-isopentenyl-2-methylthioadenine (2 ms6 iA),N⁶-methyladenine, guanine (G), isoguanine, N²-dimethylguanine (dmG),7-methylguanine (7 mG), 2-thiopyrimidine, 6-thioguanine (6 sG),hypoxanthine and O⁶-methylguanine; 7-deaza-purines such as7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidinessuch as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),4-thiothymine (4 sT), 5,6-dihydrothymine, O⁴-methylthymine, uracil (U),4-thiouracil (4 sU) and 5,6-dihydrouracil (dihydrouracil; D); indolessuch as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base(Y); as well as methylated, glycosylated, and acylated base moieties;and the like. Additional exemplary bases can be found in Fasman, 1989,in: Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fla., and the references cited therein.

Examples of nucleotides include ribonucleotides, deoxyribonucleotides,modified ribonucleotides, modified deoxyribonucleotides,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, peptide nucleotides, modified peptidenucleotides, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, and any variants of the foregoing.

Phosphate Groups

The nucleotides can optionally include phosphate groups which can belinked to the 2′, 3′ and/or 5′ position of the sugar moiety. Thephosphate groups include analogs, such as phosphoramidate,phosphorothioate, phosphorodithioate, and O-methylphophoroamiditegroups. In one embodiment, at least one of the phosphate groups can besubstituted with a fluoro and/or chloro group. The phosphate groups canbe linked to the sugar moiety by an ester or phosphoramide linkage.Typically, the nucleotide comprises three, four, five, six, seven,eight, nine, ten, or more phosphate groups linked to the 5′ position ofthe sugar moiety.

In some embodiments, the primer extension and single molecule sequencingmethods using the modified polymerases provided herein can be practicedusing any nucleotide which can be incorporated by a polymerase,including naturally-occurring or recombinant polymerases. In oneembodiment, the nucleotides can include a nucleoside linked to a chainof 1-10 phosphorus atoms. The nucleoside can include a base (or baseanalog) linked to a sugar (or sugar analog). The phosphorus chain can belinked to the sugar, for example linked to the 5′ position of the sugar.The phosphorus chain can be linked to the sugar with an intervening O orS. In one embodiment, one or more phosphorus atoms in the chain can bepart of a phosphate group having P and O. In another embodiment, thephosphorus atoms in the chain can be linked together with intervening O,NH, S, methylene, substituted methylene, ethylene, substituted ethylene,CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH₃, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. In the phosphorus chain, phosphorus atoms with anintervening atom other than O can be a substituted phosphate group. Someexamples of nucleotides are described in Xu, U.S. Pat. No. 7,405,281.

In some embodiments, the nucleotide is a dye-labeled nucleotide thatcomprises a polyphosphate chain and a dye moiety linked to the terminalphosphate group. In some embodiments, the dye-labeled nucleotidecomprises a dye moiety linked to the terminal phosphate through an alkyllinker. Optionally, the linker comprises a 6-carbon chain and has areactive amine group, and the dye moiety is linked to the terminalphosphate bond though a covalent bond formed with the amine group of thelinker. In some embodiments, the polyphosphate chain comprises 4, 5, 6,7, 8, 9, 10 or more phosphates. One exemplary dye-labeled nucleotidethat can be used in the disclosed methods and systems has the generalstructure shown in FIG. 11. This structure includes a sugar bonded to ahexaphosphate chain at the 5′ carbon position, and to a nucleotide base(denoted as “N”). The terminal phosphate group of the hexaphosphate islinked to a 6-carbon linker, and the other end of the 6-carbon linker isattached to a dye moiety (denoted as “dye”), typically through an amidebond. In some embodiments, the dye moiety can optionally comprise anyone or more of the following dyes: rhodols; resorufins; coumarins;xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles;stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones;eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), includingCy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS andDYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633,DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701,DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE; TEXASRED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes includingBODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610 (N-succinimidyl ester),ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXAFLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXAFLUOR 680 (Molecular Probes); DDAO(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any derivativesthereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes(LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) andIRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes(Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes(Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED(Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED(Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED(phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIGHARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al., 2005Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 andCF555 (Biotium).

In some embodiments, such dye-labeled nucleotides can be used to assayfor the nucleotide incorporation kinetics of a particular polymeraseaccording to the procedures described herein (see, e.g., Example 12).

Non-Hydrolyzable Nucleotides

The nucleotide binding and nucleotide incorporation methods can bepracticed using incorporatable nucleotides and non-hydrolyzablenucleotides. In the presence of the incorporatable nucleotides (e.g.,labeled), the non-hydrolyzable nucleotides (e.g., non-labeled) cancompete for the polymerase binding site to permit distinction betweenthe complementary and non-complementary nucleotides, or fordistinguishing between productive and non-productive binding events. Inthe nucleotide incorporation reaction, the presence of thenon-hydrolyzable nucleotides can alter the length of time, frequency,and/or duration of the binding of the labeled incorporatablenucleotides.

The non-hydrolyzable nucleotides can be non-labeled or can be linked toa reporter moiety (e.g., energy transfer moiety). The labelednon-hydrolyzable nucleotides can be linked to a reporter moiety at anyposition, such as the sugar, base, or any phosphate (or substitutedphosphate group). For example, the non-hydrolyzable nucleotides can havethe general structure:R₁₁—(—P)_(n)—S—B

Where B can be a base moiety, such as a hetero cyclic base whichincludes substituted or unsubstituted nitrogen-containing heteroaromaticring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, orglucosyl group. Where n can be 1-10, or more. Where P can be one or moresubstituted or unsubstituted phosphate or phosphonate groups. Where R₁₁,if included, can be a reporter moiety (e.g., a fluorescent dye). In oneembodiment, the non-hydrolyzable nucleotide having multiple phosphate orphosphonate groups, the linkage between the phosphate or phosphonategroups can be non-hydrolyzable by the polymerase. The non-hydrolyzablelinkages include, but are not limited to, amino, alkyl, methyl, and thiogroups. Non-hydrolyzable nucleotide tetraphosphates having alpha-thio oralpha boreno substitutions having been described (Rank, U.S. publishedpatent application No. 2008/0108082; and Gelfand, U.S. published patentapplication No. 2008/0293071).

The phosphate or phosphonate portion of the non-hydrolyzable nucleotidecan have the general structure:

Where B can be a base moiety and S can be a sugar moiety. Where any oneof the R₁-R₇ groups can render the nucleotide non-hydrolyzable by apolymerase. Where the sugar C5 position can be CH₂, CH₂O, CH═, CHR, orCH₂CH₂. Where the R₁ group can be O, S, CH═, CH(CN), or NH. Where theR₂, R₃, and R₄, groups can independently be O, BH₃, or SH. Where the R₅and R₆ groups can independently be an amino, alkyl, methyl, thio group,or CHF, CF₂, CHBr, CCl₂, O—O, or —C≡C—. Where the R₇ group can beoxygen, or one or more additional phosphate or phosphonate groups, orcan be a reporter moiety. Where R₈ can be SH, BH₃, CH₃, NH₂, or a phenylgroup or phenyl ring. Where R₉ can be SH. Where R₁₀ can be CH₃,N₃CH₂CH₂, NH₂, ANS, N₃, MeO, SH, Ph, F, PhNH, PhO, or RS (where Ph canbe a phenyl group or phenyl ring, and F can be a fluorine atom orgroup). The substituted groups can be in the S or R configuration.

The non-hydrolyzable nucleotides can be alpha-phosphate modifiednucleotides, alpha-beta nucleotides, beta-phosphate modifiednucleotides, beta-gamma nucleotides, gamma-phosphate modifiednucleotides, caged nucleotides, or di-nucleotides.

Many examples of non-hydrolyzable nucleotides are known (Rienitz 1985Nucleic Acids Research 13:5685-5695), including commercially-availableones from Jena Bioscience (Jena, Germany).

In some embodiments, the nucleotide comprises a releasable label and/ora releasable blocking group that can be removed via suitable means priorto incorporation of the next nucleotide by the polymerase into the newlysynthesized strand. The use of releasably labeled nucleotides whereinthe label can be cleaved and removed via suitable means have beendescribed, for example, in U.S. Pub. Nos. US2005/0244827 andUS2004/0244827, as well as U.S. Pat. Nos. 7,345,159; 6,664,079;7,345,159; and 7,223,568.

In some embodiments, the nucleotide is a nucleotide analog that iscapable of acting as a reversible terminator of nucleic acid synthesis.Typically, reversible terminators can be incorporated by a polymeraseonto the end of an extending nucleic acid molecule, but then “terminate”further synthesis by blocking further addition of nucleotides. In someembodiments, this “termination” capability can be manipulated byadjusting the reaction conditions and/or by suitable treatment. Theability to terminate can result from the presence of a moiety or group,typically named a “blocking” group, which is linked to the nucleotide.In some embodiments, the ability of the nucleotide analog to terminatenucleic acid synthesis can be eliminated through physical removal,cleavage, structural modification or disruption of the blocking group.The blocking group can be attached to any portion of the nucleotideanalog including, for example, a base moiety, sugar moiety or phosphatemoiety. The blocking group can be attached to the nucleotide analog viaa linker. The linkage between the blocking group and the nucleotideanalog can be a photocleavable, chemically cleavable, enzymaticallycleavable, thermocleavable (i.e., cleavable upon adjustment oftemperature) or pH-sensitive linkage. In some embodiments, the label(which is linked to the nucleotide) is the blocking group.

In some embodiments, the reversible terminator further comprises a labelor tag that facilitates detection of nucleotide analog. The label can bea fluorescent label. In some embodiments, the label can also be removedvia suitable treatment. In some embodiments, the label is released fromthe nucleotide analog during incorporation of the nucleotide analog intothe extending nucleic acid molecule. Alternatively, the label becomesincorporated into the extending nucleic acid molecule and is thenremoved via suitable treatment. In some embodiments, the label isattached to the nucleotide analog via a cleavable linkage. The cleavablelinkage can be a photocleavable, chemically cleavable, enzymaticallycleavable, thermocleavable (i.e., cleavable upon adjustment oftemperature) or pH-sensitive linkage.

The removal of the blocking group can be accomplished in a variety ofways. In some embodiments, the blocking group is attached to thenucleotide analog via a photocleavable linkage and can be removed fromthe nucleotide analog via exposure to photocleaving radiation. In someembodiments, the linkage is a chemically or enzymatically cleavablelinkage. In some embodiments, the linkage can be disrupted by varyingreaction conditions, e.g., pH, temperature, concentrations of divalentcations, etc.

Non-limiting examples of suitable reversible terminators include, interalia, nucleotide base-labeled nucleotide analogs comprising one or moreblocking groups attached to 3′ hydroxyl group, the base moiety or aphosphate group. For example, the nucleotide analog can comprise anazidomethyl group linked to the 3′ hydroxyl group and a fluorescentlabel linked to the base of the nucleotide analog. In some embodiments,the reversible terminator can comprise one or more blocking groupsattached to the phosphate group. In some embodiments, the nucleotideanalog can comprise a blocking group and a label. In some embodiments,both the blocking group and the label can be linked to the base moiety,while the 3′ hydroxyl group is not modified. In some embodiments, theblocking group can be a photocleavable group linked to the base of thenucleotide analog. See, e.g., U.S. Publication No. 2008/0132692,published Jun. 5, 2008. Further examples of nucleotides comprisingextension blocking groups and methods of their use in polymerase-basedapplications can be found, for example, in U.S. Pat. No. 7,078,499issued Jul. 18, 2006; as well as in U.S. Published Application Nos.2004/0048300 published Mar. 11, 2004; 2008/0132692 published Jun. 5,2008; 2009/0081686, published Mar. 26, 2009; and 2008/0131952, publishedJun. 5, 2008; Tsien, WO/1991/006678; Stemple, U.S. Pat. No. 7,270,951,Balasubramanian, U.S. Pat. No. 7,427,673; Milton, U.S. Pat. No.7,541,444.

In some embodiments, the nucleotide analog comprises a cleavable labellinked to the base. In some embodiments, the blocking group and thelabel can be removed via the same cleavage treatment. See, e.g., U.S.Pat. No. 7,553,949, issued Jun. 30, 2009. Alternatively, differenttreatments can be required to remove the blocking group and the label.In some embodiments, the label of the reversible terminator correlateswith the base identity of the nucleotide analog. In some embodiments,each reversible terminator is added sequentially to the polymerasereaction; alternatively, different kinds of reversible terminators canbe present simultaneously in the reaction mixture.

In some embodiments, the blocking group is linked to the 2′ hydroxylgroup of the sugar moiety. See, e.g., U.S. Pat. No. 7,553,949, issuedJun. 30, 2009.

In some embodiments, the reversible terminator can comprise more thanone blocking group. In some embodiments, these multiple blocking groupsmay function cooperatively by enhancing the termination efficiency ofthe nucleotide analog. In one exemplary embodiment, the nucleotideanalog comprises a blocking group linked to the base moiety, whileanother group linked to the terminal phosphate group further suppressesthe incorporation of a nucleotide analog onto the free 3′ hydroxylgroup. See, e.g., U.S. patent application Ser. No. 12/355,487, filedJan. 16, 2009.

Typically, the modified polymerases of the present disclosure can beused to sequence one or more nucleic acid molecules of interest usingreversible terminators. In an exemplary method, the reversibleterminator is incorporated in a template-dependent manner onto the 3′end of an extending nucleic acid molecule by a modified polymerase. Theincorporated reversible terminator is detected and identified; and theblocking group of the reversible terminator is then removed. In someembodiments, the unincorporated reversible terminators can be washedaway; in some embodiments, it is not necessary to wash or otherwiseremove the unincorporated reversible terminators prior to detection,identification or subsequent extension of the extending nucleic acidmolecule. In some embodiments, incorporation of the reversibleterminator onto the end of a nucleic acid molecule can involve theformation of a covalent bond between the reversible terminator and thenucleotide moiety at the 3′ end of the nucleic acid molecule.Alternatively, incorporation of reversible terminator onto the end of anucleic acid molecule will not involve formation of any covalent bondbetween the reversible terminator and the nucleotide moiety at the 3′end of the nucleic acid molecule; instead, the reversible terminator isbound in a template-dependent fashion and positioned within the activesite of the polymerase until the blocking group is cleaved or otherwiseremoved, following which the remaining portion of the reversibleterminator can remain as a portion of the extending nucleic acidmolecule or alternatively will also dissociate from the polymeraseactive site and diffuse away.

“Nanoparticle” may refer to any particle with at least one majordimension in the nanosize range. In general, nanoparticles can be madefrom any suitable metal (e.g., noble metals, semiconductors, etc.)and/or non-metal atoms. Nanoparticles can have different shapes, each ofwhich can have distinctive properties including spatial distribution ofthe surface charge; orientation dependence of polarization of theincident light wave; and spatial extent of the electric field. Theshapes include, but are not limited to: spheres, rods, discs, triangles,nanorings, nanoshells, tetrapods, nanowires, etc.

In one embodiment, the nanoparticle can be a core/shell nanoparticlewhich typically comprises a core nanoparticle surrounded by at least oneshell. For example, the core/shell nanoparticle can be surrounded by aninner and outer shell. In another embodiment, the nanoparticle is a corenanoparticle which has a core but no surrounding shell. The outmostsurface of the core or shell can be coated with tightly associatedligands which are not removed by ordinary solvation.

Examples of a nanoparticle include a nanocrystal, such as a core/shellnanocrystal, plus any associated organic ligands (which are not removedby ordinary solvation) or other materials which may coat the surface ofthe nanocrystal. In one embodiment, a nanoparticle has at least onemajor dimension ranging from about 1 to about 1000 nm. In otherembodiments, a nanoparticle has at least one major dimension rangingfrom about 1 to about 20 nm, about 1 to about 15 nm, about 1 to about 10nm or about 1 to 5 nm.

In some embodiments, a nanoparticle can have a layer of ligands on itssurface which can further be cross-linked to each other. In someembodiments, a nanoparticle can have other or additional surfacecoatings which can modify the properties of the particle, for example,increasing or decreasing solubility in water or other solvents. Suchlayers on the surface are included in the term ‘nanoparticle.’

In one embodiment, nanoparticle can refer to a nanocrystal having acrystalline core, or to a core/shell nanocrystal, and may be about 1 nmto about 100 nm in its largest dimension, about 1 nm to about 20 nm,about 1 nm to about 15 nm, about 1 nm to about 10 nm or preferably about5 nm to about 10 nm in its largest dimension. Small nanoparticles aretypically less than about 20 nm in their largest dimension.

“Nanocrystal” as used herein can refer to a nanoparticle made out of aninorganic substance that typically has an ordered crystalline structure.It can refer to a nanocrystal having a crystalline core (corenanocrystal) or to a core/shell nanocrystal.

A core nanocrystal is a nanocrystal to which no shell has been applied.Typically, it is a semiconductor nanocrystal that includes a singlesemiconductor material. It can have a homogeneous composition or itscomposition can vary with depth inside the nanocrystal.

A core/shell nanocrystal is a nanocrystal that includes a corenanocrystal and a shell disposed over the core nanocrystal. Typically,the shell is a semiconductor shell that includes a single semiconductormaterial. In some embodiments, the core and the shell of a core/shellnanocrystal are composed of different semiconductor materials, meaningthat at least one atom type of a binary semiconductor material of thecore of a core/shell is different from the atom types in the shell ofthe core/shell nanocrystal.

The semiconductor nanocrystal core can be composed of a semiconductormaterial (including binary, ternary and quaternary mixtures thereof),from: Groups II-VI of the periodic table, including ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V, including GaN,GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/orGroup IV, including Ge, Si, Pb.

The semiconductor nanocrystal shell can be composed of materials(including binary, ternary and quaternary mixtures thereof) comprising:ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP,GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP,or AlSb.

Many types of nanocrystals are known, and any suitable method for makinga nanocrystal core and applying a shell to the core may be employed.Nanocrystals can have a surface layer of ligands to protect thenanocrystal from degradation in use or during storage.

“Quantum dot” as used herein refers to a crystalline nanoparticle madefrom a material which in the bulk is a semiconductor or insulatingmaterial, which has a tunable photophysical property in the nearultraviolet (UV) to far infrared (IR) range.

“Water-soluble” or “water-dispersible” is used herein to mean the itemcan be soluble or suspendable in an aqueous-based solution, such as inwater or water-based solutions or buffer solutions, including those usedin biological or molecular detection systems as known by those skilledin the art. While water-soluble nanoparticles are not truly ‘dissolved’in the sense that term is used to describe individually solvated smallmolecules, they are solvated (via hydrogen, electrostatic or othersuitable physical/chemical bonding) and suspended in solvents which arecompatible with their outer surface layer, thus a nanoparticle which isreadily dispersed in water is considered water-soluble orwater-dispersible. A water-soluble nanoparticle can also be consideredhydrophilic, since its surface is compatible with water and with watersolubility.

“Hydrophobic nanoparticle” as used herein refers to a nanoparticle whichis readily dispersed in or dissolved in a water-immiscible solvent likehexanes, toluene, and the like. Such nanoparticles are generally notreadily dispersed in water.

“Hydrophilic” as used herein refers to a surface property of a solid, ora bulk property of a liquid, where the solid or liquid exhibits greatermiscibility or solubility in a high-dielectric medium than it does in alower dielectric medium. By way of example, a material which is moresoluble in methanol than in a hydrocarbon solvent such as decane wouldbe considered hydrophilic.

“Coordinating solvents” as used herein refers to a solvent such as TDPA,OP, TOP, TOPO, carboxylic acids, and amines, which are effective tocoordinate to the surface of a nanocrystal. ‘Coordinating solvents’ alsoinclude phosphines, phosphine oxides, phosphonic acids, phosphinicacids, amines, and carboxylic acids, which are often used in growthmedia for nanocrystals, and which form a coating or layer on thenanocrystal surface. Coordinating solvents can exclude hydrocarbonsolvents such as hexanes, toluene, hexadecane, octadecene and the like,which do not have heteroatoms that provide bonding pairs of electrons tocoordinate with the nanocrystal surface. Hydrocarbon solvents which donot contain heteroatoms such as O, S, N or P to coordinate to ananocrystal surface are referred to herein as non-coordinating solvents.Note that the term ‘solvent’ is used in its ordinary way in these terms:it refers to a medium which supports, dissolves or disperses materialsand reactions between them, but which does not ordinarily participate inor become modified by the reactions of the reactant materials. However,in certain instances, the solvent can be modified by the reactionconditions. For example, TOP may be oxidized to TOPO, or a carboxylicacid can be reduced to an alcohol.

As used herein, the term “population” refers to a plurality ofnanoparticles having similar physical and/or optical properties.‘Population’ can refer to a solution or structure with more than onenanoparticle at a concentration suitable for single molecule analysis.In some embodiments, the population can be monodisperse and can exhibitless than at least 15% rms deviation in diameter of the nanoparticles,and spectral emissions in a narrow range of no greater than about 75 nmfull width at half max (FWHM). In the context of a solution, suspension,gel, plastic, or colloidal dispersion of nanoparticles, the nature ofthe population can be further characterized by the number ofnanoparticles present, on average, within a particular volume of theliquid or solid, or the concentration. In a two-dimensional format suchas an array of nanoparticles adhered to a solid substrate, the conceptof concentration is less convenient than the related measure of particledensity, or the number of individual particles per two-dimensional area.In this case, the maximum density would typically be that obtained bypacking particles “shoulder-to-shoulder” in an array. The actual numberof particles in this case would vary due to the size of the particles—agiven array could contain a large number of small particles or a smallnumber of larger particles.

As used herein, the terms “moderate to high excitation” refers tomonochromatic illumination or excitation (e.g., laser illumination)having a high power intensity sufficiently high such that the absorbedphotons per second for a given sample is between about 200,000 and about1,600,000.

In one aspect, the nanoparticle is a semiconductor nanoparticle havingsize-dependent optical and electronic properties. For example, thenanoparticle can emit a fluorescent signal in response to excitationenergy. The spectral emission of the nanoparticle can be tunable to adesired energy by selecting the particle size, size distribution, and/orcomposition of the semiconductor nanoparticle. For example, depending onthe dimensions, the semiconductor nanoparticle can be a fluorescentnanoparticle which emits light in the UV-visible-IR spectrum. The shellmaterial can have a bandgap greater than the bandgap of the corematerial.

In one aspect, the nanoparticle is an energy transfer donor. Thenanoparticle can be excited by an electromagnetic source such as a laserbeam, multi-photon excitation, or electrical excitation. The excitationwavelength can range between about 190 to about 800 nm including allvalues and ranges there in between. In some embodiments, thenanoparticle can be excited by an energy source having a wavelength ofabout 405 nm. In other embodiments, in response to excitation, thenanoparticle can emit a fluorescent signal at about 400-800 nm, or about605 nm.

In one aspect, the nanoparticle can undergo Raman scattering whensubjected to an electromagnetic source (incident photon source) such asa laser beam. The scattered photons have a frequency that is differentfrom the frequency of the incident photons. As result, the wavelength ofthe scattered photons is different than the incident photon source. Inone embodiment, the nanoparticle can be attached to a suitable tag orlabel to enhance the detectability of the nanoparticle via Ramanspectroscopy. The associated tag can be fluorescent or nonfluorescent.Such approaches can be advantageous in avoiding problems that can arisein the context of fluorescent nanoparticles, such as photobleaching andblinking. See, e.g., Sun et al., “Surface-Enhanced Raman ScatteringBased Nonfluorescent Probe for Multiplex DNA Detection”, Anal. Chem.79(11):3981-3988 (2007).

In one aspect, the nanoparticle is comprised of a multi-shell layeredcore which is achieved by a sequential shell material depositionprocess, where one shell material is added at a time, to provide ananoparticle having a substantially uniform shell of desired thicknesswhich is substantially free of defects. The nanoparticle can be preparedby sequential, controlled addition of materials to build and/or applyinglayers of shell material to the core. See e.g., U.S. PCT ApplicationSerial No. PCT/US09/61951 which is incorporated herein by reference asif set forth in full.

In another aspect, a method is provided for making a nanoparticlecomprising a core and a layered shell, where the shell comprises atleast one inner shell layer and at least one outer shell layer. Themethod comprises the steps: (a) providing a mixture comprising a core,at least one coordinating solvent; (b) heating the mixture to atemperature suitable for formation of an inner shell layer; (c) adding afirst inner shell precursor alternately with a second inner shellprecursor in layer additions, to form an inner shell layer which is adesired number of layers thick; (d) heating the mixture to a temperaturesuitable for formation of an outer shell layer; and (e) adding a firstouter shell precursor alternately with a second outer shell precursor inlayer additions, to form an outer shell layer which is a desired numberof layers thick. In one embodiment, if the coordinating solvent of (a)is not amine, the method further comprises an amine in (a).

In one aspect, at least one coordinating solvent comprises atrialkylphosphine, a trialkylphosphine oxide, phosphonic acid, or amixture of these. In another aspect, at least one coordinating solventcomprises trioctylphosphine (TOP), trioctylphosphine oxide (TOPO),tetradecylphosphonic acid (TDPA), or a mixture of these. In yet anotheraspect, the coordinating solvent comprises a primary or secondary amine,for example, decylamine, hexadecylamine, or dioctylamine.

In one aspect, the nanoparticle comprises a core comprising CdSe. Inanother aspect, the nanoparticle shell can comprise YZ wherein Y is Cdor Zn, and Z is S, or Se. In one embodiment, at least one inner shelllayer comprises CdS, and the at least one outer shell layer comprisesZnS.

In one aspect, the first inner shell precursor is Cd(OAc)₂ and thesecond inner shell precursor is bis(trimethylsilyl)sulfide (TMS₂S). Inother aspects, the first and second inner shell precursors are added asa solution in trioctylphosphine (TOP). In other aspects, the first outershell precursor is diethylzinc (Et₂Zn) and the second inner shellprecursor is dimethyl zinc (TMS₂S). Sometimes, the first and secondouter shell precursors are added as a solution in trioctylphosphine(TOP).

In one aspect, the nanoparticle can have ligands which coat the surface.The ligand coating can comprise any suitable compound(s) which providesurface functionality (e.g., changing physicochemical properties,permitting binding and/or other interaction with a biomolecule, etc.).In some embodiments, the disclosed nanoparticle has a surface ligandcoating (in direct contact with the external shell layer) that addsvarious functionalities which facilitate it being water-dispersible orsoluble in aqueous solutions. There are a number of suitable surfacecoatings which can be employed to permit aqueous dispersibility of thedescribed nanoparticle. For example, the nanoparticle(s) disclosedherein can comprise a core/shell nanocrystal which is coated directly orindirectly with lipids, phospholipids, fatty acids, polynucleic acids,polyethylene glycol (PEG), primary antibodies, secondary antibodies,antibody fragments, protein or nucleic acid based aptamers, biotin,streptavidin, proteins, peptides, small organic molecules (e.g.,ligands), organic or inorganic dyes, precious or noble metal clusters.Specific examples of ligand coatings can include, but are not limitedto, amphiphilic polymer (AMP), bidentate thiols (i.e., DHLA), tridentatethiols, dipeptides, functionalized organophosphorous compounds (e.g.,phosphonic acids, phosphinic acids), etc.

Non-Blinking Nanoparticles

Provided herein are nanoparticles which exhibit modulated, reduced, orno intermittent (e.g., continuous, non-blinking) fluorescence.

In one aspect, the nanoparticle or populations thereof exhibitmodulated, reduced or non-detectable intermittent (e.g., continuous,etc.) fluorescence properties. The nanoparticles can have a stochasticblinking profile in a timescale which is shifted to very rapid blinkingor very slow or infrequent blinking relative to a nanoparticlepreviously described in the art (conventional nanoparticles aredescribed in the art as having on-time fractions of <0.2 in the best ofconditions examined). For example, the nanoparticles may blink on andoff on a timescale which is too rapid to be detected under the methodsemployed to study this behavior.

In one aspect the nanoparticle or populations thereof are photostable.The nanoparticles can exhibit a reduced or no photobleaching with longexposure to moderate to high intensity excitation source whilemaintaining a consistent spectral emission pattern.

In one aspect, the nanoparticle or populations thereof have aconsistently high quantum yield. For example, the nanoparticles can havea quantum yield greater than: about 10%, or about 20%, or about 30%, orabout 40%, or about 50%, or about 60%, or about 70% or about 80%.

As used herein, fluorescence (or Forster) resonance energy transfer(FRET) is a process by which a fluorophore (the donor) in an excitedstate transfers its energy to a proximal molecule (the acceptor) bynonradiative dipole-dipole interaction (Forster, T. “IntermolecularEnergy Migration and Fluorescence”, Ann. Phys., 2:55-75, 1948; Lakowicz,J. R., Principles of Fluorescence Spectroscopy, 2nd ed. Plenum, N.Y.367-394, 1999).

FRET efficiency (E) can be defined as the quantum yield of the energytransfer transition, i.e. the fraction of energy transfer eventoccurring per donor excitation event. It is a direct measure of thefraction of photon energy absorbed by the donor which is transferred toan acceptor, as expressed in Equation 1: E=k_(ET)/k_(f)+k_(ET)+Σk_(i)where k_(ET) is the rate of energy transfer, k_(f) the radiative decayrate and the k_(i) are the rate constants of any other de-excitationpathway.

FRET efficiency E generally depends on the inverse of the sixth power ofthe distance r (nm) between the two fluorophores (i.e., donor andacceptor pair), as expressed in Equation 2: E=1/1+(r/R₀)⁶.

The distance where FRET efficiency is at 50% is termed R₀, also know asthe Forster distance. R₀ can be unique for each donor-acceptorcombination and can range from between about 5 nm to about 10 nm.Therefore, the FRET efficiency of a donor (i.e., nanoparticle) describesthe maximum theoretical fraction of photon energy which is absorbed bythe donor (i.e., nanoparticle) and which can then be transferred to atypical organic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).

In some embodiments, the disclosed nanoparticles are relatively small(i.e., <15 nm) and thus may be particularly well suited to be used as adonor or an acceptor in a FRET reaction. That is, some embodiments ofthe disclosed nanoparticles exhibit higher FRET efficiency thanconventional nanoparticles and thus are excellent partners (e.g., donorsor acceptors) in a FRET reaction.

“Quantum yield” as used herein refers to the emission efficiency of agiven fluorophore assessed by the number of times which a defined event,e.g., light emission, occurs per photon absorbed by the system. In otherwords, a higher quantum yield indicates greater efficiency and thusgreater brightness of the described nanoparticle or populations thereof.

Any suitable method can be used to measure quantum yield. In oneexample, quantum yield can be obtained using standard methods such asthose described in Casper et al (Casper, J. V.; Meyer, T. J. J. Am.Chem. Soc. 1983, 105, 5583) and can be analyzed relative to knownfluorophores chosen as appropriate for maximal overlap between standardemission and sample emission (e.g., fluorescein, Rhodamine 6G, Rhodamine101). Dilute solutions of the standard and sample can be matched ornearly matched in optical density prior to acquisition of absorbance andemission spectra for both. The emission quantum yield (ϕ_(em)) then canbe determined according to Equation 3:

$\phi_{em} = {{\phi_{em}^{\prime}( \frac{I}{I^{\prime}} )}( \frac{A^{\prime}}{A} )}$where A and A′ are the absorbances at the excitation wavelength for thesample and the standard respectively and I and I′ are the integratedemission intensities for the sample and standard respectively. In thiscase ϕ_(em)′ can be the agreed upon quantum yield for the standard.

Disclosed herein are fluorescent nanoparticles with superior and robustproperties which significantly expand the applications in whichnanoparticles are useful. These nanoparticles are superior andsurprisingly robust in that they are simultaneously stable, bright, andsensitive to environmental stimuli. Moreover, the disclosednanoparticles have limited or no detectable blinking (i.e., where thenanoparticle emits light non-intermittently when subject to excitation),are highly photostable, have a consistently high quantum yield, aresmall (e.g., ≤20 nm) and can act as a donor which undergoes FRET with asuitable acceptor moiety (e.g., fluorescent dyes, etc.). Thephotostability of these nanoparticles is reflected in their exhibitingreduced or no photobleaching (i.e., fading) behavior when subjected tomoderate to high intensity excitation for at least about 20 minutes.Additionally, the particles can remain substantially free fromphoto-induced color shifting.

Put another way, the nanoparticles can maintain a consistent spectralemission pattern (i.e., maintain the ability to fluoresce) even whenexposed to a large quantity of photons (i.e., moderate to high intensityexcitation) for a long period of time. This unique combination ofcharacteristics makes these types of nanoparticles sensitive tools forsingle molecule analysis and other sensitive high throughputapplications. Moreover, these properties make the nanoparticlesparticularly well suited for use as highly efficient donor fluorophoresin energy transfer reactions such as FRET reactions (i.e., high FRETefficiency) or other reactions as well as applications which require orare enhanced by greater response to the environment.

Without being bound to a particular theory, blinking or fluorescenceintermittency may arise during the nanoparticle charging process when anelectron is temporarily lost to the surrounding matrix (Auger ejectionor charge tunneling) or captured to surface-related trap states. Thenanoparticle is “on” or fluorescing when all of the electrons are intactand the particle is “neutral” and the particle is “off” or dark when theelectron is lost and the particle is temporarily (or in some casespermanently) charged. It is important to note that the completesuppression of blinking may not necessarily be required and in someinstances may not be desirable. Blinking which occurs on a timescalemuch shorter or much longer than the interrogation period for aparticular assay has relatively little impact on the performance of thesystem. Thus, nanoparticles and nanoparticle populations havingmodulated blinking properties, where blinking occurs on a very short orvery fast timescale relative to the assay interrogation periods are alsouseful and fall within the scope of the present disclosure. Localizationof timescale or simply pushing timescale to one side (e.g., to where theblinking is undetectable within the assay system) can providesubstantial benefit in application development.

The blinking behavior of the nanoparticles described herein can beanalyzed and characterized by any suitable number of parameters usingsuitable methodologies. The probability distribution function of the“on” and “off” blinking time durations (i.e., blinking behavior) can bedetermined using the form of an inverse power law. A value, alpha (α)can be calculated, wherein α □ represents an exponent in the power law.As the percentage of the population which is non-blinking increases, thevalue of α_(on) theoretically approaches zero. In conventionalnanoparticle populations previously described, α_(on) typically rangesfrom about 1.5 to about 2.5, under moderate to high excitation energy.

Most alpha calculations can use a predetermined threshold to determinethe “on” and “off” values of alpha-on and alpha-off (i.e., α_(on) andα_(off)). Typically, an alpha estimator which calculates the on/offthreshold for each dot individually can be employed. The data can berepresented by a plot of signal versus frequency, and typically appearsas a series of Gaussian distributions around the “off state” and one ormore “on states.” A log-log plot of frequency versus time for eachperiod of time that the dot is “on” provides a straight line having aslope of α_(on). The value of alpha-off (α_(off)) can be similarlydetermined.

In a specific example (the “TIRF example”), the fluorescentintermittency measurements can be made using a Total Internal ReflectionFluorescence (TIRF) microscope fitted with a 60× oil immersion objectivelens, using a dual view with a longpass filter on the acceptor side anda bandpass filter on the donor side. Using the TIRF setup, thenanoparticles were imaged at 30 Hz (33 ms), typically for 5 minutes, toproduce a movie showing the time and intensity of the emitted light foreach individual spot (corresponding to a single particle) within abinned frame which was 33 ms long; the intensity for each binned framecan be integrated. Each data set can be manually analyzed dot-by-dot,and aggregates and other artifacts were excluded. From the editedresults, the following parameters can be calculated: alpha-on(“α_(on)”); alpha-off (“α_(off)”); the percent on; longest on/longestoff; overlap scores; and the median values for each of these parameters.

In some aspects, provided herein is a nanoparticle or population thereofwhich has an α_(on) of less than about 1.5, α_(on) of less than about1.4, α_(on) of less than about 1.3, α_(on) of less than about 1.2, or anα_(on) of less than about 1.1, under moderate to high excitation energy.In some embodiments, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 98%, at least about 99% or more of the population has anα_(on) of less than about 1.5, α_(on) of less than about 1.4, α_(on) ofless than about 1.3, α_(on) of less than about 1.2, or α_(on) of lessthan about 1.1 for the time observed, under moderate to high excitationenergy. The observation time can be at least about 5 minutes, at leastabout 10 minutes, at least about 15 minutes, at least about 30 minutes,at least about 45 minutes, at least about 60 minutes, at least about 90minutes, at least about 120 minutes or more under moderate to highexcitation energy. Compositions comprising such a nanoparticle andpopulations thereof also are contemplated.

In some aspects, provided herein is a nanoparticle or a populationthereof having a stochastic blinking profile which is eitherundetectable or rare (e.g., no more than 1-2 events during theinterrogation period) over an observed timescale. In this case,“undetectable” encompasses the situation in which evidence might existfor ultra-fast blinking on a timescale which is faster than the binningtimescale (e.g., dimming and brightening from bin to bin) but there areno “off” events persisting for longer than the bin time. Therefore, insome embodiments, a nanoparticle or population thereof has a stochasticblinking profile which is undetectable for at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99% or more of the time observed, under moderate to highexcitation energy. In other embodiments, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 98%, at least about 99% or more ofthe individual nanoparticles in a population have a stochastic blinkingon a timescale which is undetectable for the time observed, undermoderate to high excitation energy. The timescale can be at least about5 minutes, at least about 10 minutes, at least about 15 minutes, atleast about 30 minutes, at least about 45 minutes, at least about 60minutes, at least about 90 minutes, at least about 120 minutes or moreunder moderate to high excitation energy.

In some aspects, the longest on and longest off values can relate to thelongest period of time a nanoparticle is observed to be in either the“on” or the “off” state. In particular, the longest on value can beimportant to determining the length of time and amount of data which maybe measured in a particular assay.

Thus, the blinking characteristics of the nanoparticles herein can alsobe characterized by their on-time fraction, which represents the (totalon-time)/(total experiment time). Under the TIRF example disclosedherein, the total on time can be determined by the total number offrames “on” multiplied by 33 ms, and the total experiment time is 5minutes. For example, the blinking properties of the disclosednanoparticles or populations thereof can be determined under continuousirradiation conditions using a 405 nm laser with an intensity of about 1watt per cm² during an experimental window of at least 5 minutes.

On-time fractions can be used to characterize the blinking behavior of asingle nanoparticle or of a population of nanoparticles. It is importantto note that the on-time fraction for a particular nanoparticle orpopulation of nanoparticles is a function of the specific conditionsunder which the percent of blinking or “non-blinking” nanoparticles isdetermined.

In some aspects, provided herein is a nanoparticle or population thereofhaving an on-time fraction of at least about 0.50, at least about 0.60,at least about 0.70, at least about 0.75, at least about 0.80, at leastabout 0.85, at least about 0.90, at least about 0.95, at least about0.96, at least about 0.97, at least about 0.98, or at least about 0.99or more, under moderate to high excitation energy. In some embodiments,a nanoparticle or populations thereof having a percent on-time of about98%, about 99% (i.e., on-time fraction of about 0.99) can be consideredto be “non-blinking,” under moderate to high excitation energy. In someembodiments, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more of the individual nanoparticlesin a population of nanoparticles can have an on-time fraction of atleast about 0.50, at least about 0.60, at least about 0.70, at leastabout 0.75, at least about 0.80, at least about 0.85, at least about0.90, at least about 0.95, at least about 0.96, at least about 0.97, atleast about 0.98, or at least about 0.99 or more, under moderate to highexcitation energy. The on-times of the nanoparticles are typically forat least about 5 minutes, at least about 10 minutes, at least about 15minutes, at least about 20 minutes, at least about 30 minutes, at leastabout 45 minutes, at least about 60 minutes, at least about 70 minutes,at least about 80 minutes, at least about 90 minutes, at least about 120minutes under moderate to high intensity excitation of the nanoparticleor nanoparticle population. Under one set of conditions, continuousirradiation with 405 nm laser with an approximate intensity of 1 wattper cm² was used to determine the stochastic blinking profile.

In some embodiments, nanoparticles which have a stochastic (i.e.,random) blinking profile in a timescale which shifts from very rapidblinking or very slow/infrequent blinking (relative to a nanoparticlepreviously described in the art) can be considered to have modulatedblinking properties. In some embodiments, these nanoparticles may blinkon and off on a timescale which is too rapid to be detected under themethods employed to study this behavior. Thus, certain nanoparticles caneffectively appear to be “always on” or to have on-time fractions ofabout 0.99, when in fact they flicker on and off at a rate too fast ortoo slow to be detected. Such flickering has relatively little impact onthe performance of a system, and for practical purposes suchnanoparticles can be considered to be non-blinking.

In some instances, the disclosed nanoparticles and populations thereofare not observed to blink off under the analysis conditions, and suchparticles can be assessed as “always on” (e.g., non-blinking). Thepercent of usable dots which are “always on” can be a useful way tocompare nanoparticles or populations of nanoparticles. However, adetermination of “always on” may mean that the “off” time wasinsufficient to provide enough a signal gap for accurate determinationand thus the value in the regime of particles is insufficient tocalculate. Even these “non-blinking” nanoparticles may flicker on andoff on a timescale which is not detected under the conditions used toassess blinking. For example, certain particles may blink on a timescalewhich is too fast to be detected, or they may blink very rarely, and, insome embodiments, such particles may also be considered to be“always-on” or non-blinking, as the terms are used herein.

In one aspect, provided herein is a nanoparticle or population thereofwhich demonstrate some fluctuation in fluorescence intensity. In someembodiments, the change in fluorescence intensity for the nanoparticleis less than about 5%, less than about 10%, less than about 20%, or lessthan about 25% of the nanoparticle or populations thereof at itsgreatest intensity, under moderate to high excitation energy. In someembodiments, such changes in fluorescence intensity of less than about5%, less than about 10%, less than about 20%, or less than about 25% ofthe highest intensity can occur in at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99% of the nanoparticles in the population, under moderate to highexcitation energy.

In some aspects, the nanoparticles with modulated, reduced or nointermittent (e.g., continuous, non-blinking) fluorescence providedherein can comprise of a core and a layered gradient shell. In someembodiments, the nanoparticle(s) disclosed herein can be comprised of ananocrystal core (e.g., CdSe, etc.), at least one inner (intermediate)shell layer (e.g., CdS, etc.), and at least one outer (external) shelllayer (e.g., ZnS, etc.). In some embodiments, the inner and/or outershell layers are each comprised of two or more discrete monolayers ofthe same material. In some embodiments, the largest dimension of thedisclosed nanoparticle(s) is less than about 15 nm. See for example, PCTApplication Serial No. PCT US/09/61951. See also PCT/US09/061951 andPCT/US09/061953 both filed on Oct. 23, 2009.

As discussed previously, the disclosed nanoparticles may be particularlywell suited for use as a donor or acceptor which undergoes FRET with asuitable complementary partner (donor or acceptor). A “FRET capable”nanoparticle refers to a nanoparticle which can undergo a measurableFRET energy transfer event with a donor or an acceptor moiety. In someembodiments, a FRET capable nanoparticle is one which has at least about25% efficiency in a FRET reaction.

Thus, in one aspect, a FRET capable fluorescent nanoparticle orpopulation thereof with modulated, reduced or non intermittent (e.g.,continuous, etc.) fluorescence is provided. In some embodiments, thenanoparticle is the donor in a FRET reaction. In some embodiments, thenanoparticle is the acceptor in the FRET reaction.

In some embodiments, the FRET capable non-blinking fluorescentnanoparticle(s) disclosed herein can comprise a core and a layeredgradient shell. In some embodiments, the FRET capable non-blinkingnanoparticle(s) disclosed herein can be comprised of a nanocrystal core(e.g., CdSe, etc.), at least one inner (intermediate) shell layer (e.g.,CdS, etc.), and at least one outer (external) shell layer (e.g., ZnS,etc.). In some embodiments, the inner and/or outer shell layers are eachcomprised of two or more discrete monolayers of the same material. Insome embodiments, the largest dimension of the disclosed FRET capablenanoparticle(s) is less than about 15 nm.

In some embodiments, the nanoparticle or population thereof has a FRETefficiency of at least about 20%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or greater.

In some embodiments, at least about 30%, at least about 40%, at leastabout 50%, at least about 60% at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 98%, at least about99% or more of the individual nanoparticles in the population have aFRET efficiency of at least about 20%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99% or more.

In some embodiments, the FRET efficiency of the disclosed nanoparticleor population thereof can be maintained for at least about the first10%, at least about the first 20%, at least about the first 30%, atleast about the first 40%, at least about the first 50%, at least aboutthe first 60%, at least about the first 70%, at least about the first80%, at least about the first 90% or more of the total emitted photonsunder conditions of moderate to high excitation.

As discussed above, the nanoparticle(s) provided herein can beconsidered to be surprisingly photostable. In particular, thenanoparticle and populations described herein can be photostable over anextended period of time while maintaining the ability to effectivelyparticipate in energy transfer (i.e., FRET) reactions. The disclosednanoparticles can be stable under high intensity conditions involvingprolonged or continuous irradiation over an extended period of time froma moderate to high excitation source.

Thus, in one aspect, provided herein is a non-blinking fluorescentnanoparticle and population thereof which is photostable.

In some embodiments, the disclosed photostable nanoparticle andpopulation thereof can have an emitted light or energy intensitysustained for at least about 10 minutes and does not decrease by morethan about 20% of maximal intensity achieved during that time. Further,these nanoparticles and populations thereof can have a wavelengthspectrum of emitted light which does not change more than about 10% uponprolonged or continuous exposure to an appropriate energy source (e.g.irradiation).

In one embodiment, the photostable nanoparticles disclosed herein canremain photostable under moderate to high intensity excitation from atleast about 10 minutes to about 2 hours. In another embodiment, thephotostable nanoparticles disclosed herein can remain photostable undermoderate to high intensity excitation from at least about 10 minutes toabout 10 hours. In still another embodiment, the photostablenanoparticles disclosed herein can remain photostable under moderate tohigh from about 10 minutes to about 48 hours. However, it should beappreciated, that these are just example photostable times for thedisclosed nanoparticles, in practice the nanoparticles can remainphotostable for longer periods of time depending on the particularapplication.

It should be appreciated that nanoparticles which are photostable overlonger timescales in combination with moderate to high excitation energysources are well suited for more sensitive and broad-rangingapplications such as the real-time monitoring of single moleculesinvolving FRET. That is, the nanoparticle and population thereofdescribed herein can be photostable over an extended period of timewhile maintaining the ability to effectively participate in energytransfer (i.e., FRET) reactions, which makes the subject nanoparticlesparticularly useful for many applications involving the real-timemonitoring of single molecules. As such, in some embodiments thephotostable nanoparticles disclosed herein have FRET efficiencies of atleast about 20%.

In some embodiments, the disclosed nanoparticles are stable uponprolonged or continuous irradiation (under moderate to high excitationrate) in which they do not exhibit significant photo-bleaching on thetimescales indicated. Photobleaching can result from the photochemicaldestruction of a fluorophore (and can be characterized by thenanoparticles losing the ability to produce a fluorescent signal) by thelight exposure or excitation source used to stimulate the fluorescence.Photobleaching can complicate the observation of fluorescent moleculesin microscopy and the interpretation of energy transfer reactionsbecause the signals can be destroyed or diminished increasingly astimescales for the experiment increase or the energy intensityincreases.

Photobleaching can be assessed by measuring the intensity of the emittedlight or energy for a nanoparticle or nanoparticle population using anysuitable method. In some embodiments, the intensity of emitted light orenergy from the disclosed nanoparticle or population thereof does notdecrease by more than about 20% (and in some embodiments, not more thanabout 10%) upon prolonged or continuous irradiation (under moderate tohigh excitation rate). In some embodiments, the intensity of emittedlight from the disclosed nanoparticle or population thereof does notdecrease by more than about 20%, about 15%, about 10%, about 5% or lessupon irradiation from about 10 minutes, about 20 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2hours, about 3 hours to about 4 hours, under moderate to high excitationenergy.

In some embodiments, the photostable nanoparticles provided hereinfurther demonstrate enhanced stability in which they exhibit a reductionin or absence of spectral shifting during prolonged excitation. In theconventional nanoparticles previously described in the art, increasedexposure to an excitation source—whether via increase time orpower—results in a spectral shift of the wavelength emission wavelengthprofile of a nanoparticle and populations thereof from a longerwavelength to an increasingly shorter wavelength. Such spectral shiftingof emission wavelength represents a significant limitation as preciseresolution of emission spectra is required for applications whichrequire rapid detection, multi-color analysis, and the like. Shifting ofany significance then requires that the wavelength emissions used in anassay be sufficiently separated to permit resolution, thus reducing thenumber of colors available as well as increasing signal to noise ratioto an unacceptable level as the initial spectral profile cannot berelied upon once spectral shifting begins. Such shifting may requireshortened observation times or use of fluorophores with widely separatedemission spectra. The nanoparticles provided herein have little to nospectral shift, particularly over extended periods of excitation.

Wavelength emission spectra can be assessed by any suitable method. Forexample, spectral characteristics of nanoparticles can generally bemonitored using any suitable light-measuring or light-accumulatinginstrumentation. Examples of such instrumentation are CCD(charge-coupled device) cameras, video devices, CIT imaging, digitalcameras mounted on a fluorescent microscope, photomultipliers,fluorometers and luminometers, microscopes of various configurations,and even the human eye. The emission can be monitored continuously or atone or more discrete time points. The photostability and sensitivity ofnanoparticles allow recording of changes in electrical potential overextended periods of time.

Thus, in some embodiments, the photostable nanoparticle and populationthereof has a wavelength spectrum of emitted light which does not changemore than about 10% upon prolonged or continuous exposure to anappropriate energy source (e.g. irradiation) over about 4 minutes toabout 10 minutes, under moderate to high excitation energy. In someembodiments, the wavelength emission spectra does not change more thanabout 5%, more than about 10%, more than about 20% over 10 minutes,about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes,about 90 minutes, about 2 hours, about 3 hours to about 4 hours.

It should be appreciated that there can be various other objectiveindicia of nanoparticle photostability. For example, a nanoparticle canbe classified as photostable when the nanoparticle, under moderate tohigh excitation, emits about 1,000,000 to about 100,000,000 photons ormore preferably about 100,000,001 to about 100,000,000,000 photons oreven more preferably more than about 100,000,000,000 photons beforebecoming non-emissive (i.e., bleached).

A nanoparticle with modulated, reduced or no fluorescent intermittency(e.g., continuous, non-blinking, etc.); reduced or absent spectralshifting; low to no photobleaching; high quantum yield; and sufficientFRET efficiency can be of any suitable size. Typically, it is sized toprovide fluorescence in the UV-visible portion of the electromagneticspectrum as this range is convenient for use in monitoring biologicaland biochemical events in relevant media. The disclosed nanoparticle andpopulation thereof can have any combination of the properties describedherein.

Thus, in some embodiments the nanoparticle or population thereof hasmodulated or no blinking, are photostable (e.g., limited or nophotobleaching, limited or no spectral shift), has high quantum yield,have high FRET efficiency, has a diameter of less than about 15 nm, isspherical or substantially spherical shape, or any combination of allthese properties as described herein.

Likewise, in some embodiments, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or more of theindividual nanoparticles in a population of nanoparticles have modulatedor no blinking, are photostable (e.g., limited or no photobleaching,limited or no spectral shift), have high quantum yield, have high FRETefficiency, have diameters of less than about 15 nm, are spherical orsubstantially spherical shape, or any combination of or all of theseproperties as described herein.

In one aspect, the FRET capable, non-blinking and/or photostablenanoparticle or population thereof provided herein has a maximumdiameter of less than about 20 nm. In some embodiments, thenanoparticle(s) can be less than about 15 nm, less than about 10 nm,less than about 8 nm, less than about 6 nm, less than about 5 nm, lessthan about 4 nm, less than about 3 nm or less in its largest diameterwhen measuring the core/shell structure. Any suitable method may be usedto determine the diameter of the nanoparticle(s). The nanoparticle(s)provided herein can be grown to the desired size using any of themethods disclosed herein. In some embodiments, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or moreof the individual members of a population of nanoparticles have maximumdiameters (when measuring the core, core/shell or core/shell/ligandstructure) which are less than about 20 nm, less than about 15 nm, lessthan about 10 nm, less than about 8 nm, less than about 6 nm, less thanabout 5 nm, less than about 4 nm, less than about 3 nm or less.

The FRET capable, non-blinking and/or photostable nanoparticle(s)provided herein and populations thereof can be spherical orsubstantially spherical. In some embodiments, a substantially sphericalnanoparticle can be one where any two radius measurements do not differby more than about 10%, about 8%, about 5%, about 3% or less. In someembodiments, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more of the individual members of apopulation of nanoparticles are spherical or substantially spherical.

Nanoparticles can be synthesized in shapes of different complexity suchas spheres, rods, discs, triangles, nanorings, nanoshells, tetrapods,nanowires and so on. Each of these geometries can have distinctiveproperties: spatial distribution of the surface charge, orientationdependence of polarization of the incident light wave, and spatialextent of the electric field. In some embodiments, the nanoparticles aresubstantially spherical or spheroidal.

For embodiments where the nanoparticle is not spherical or spheroidal,e.g. rod-shaped, it may be from about 1 to about 15 nm, from about 1 nmto about 10 nm, or 1 nm to about 5 nm in its smallest dimension. In somesuch embodiments, the nanoparticles may have a smallest dimension ofabout 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm,about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 25 nm, about30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm and rangesbetween any two of these values.

The single-color preparation of the nanoparticles disclosed herein canhave individual nanoparticles which are of substantially identical sizeand shape. Thus, in some embodiments, the size and shape between theindividual nanoparticles in a population of nanoparticles vary by nomore than about 20%, no more than about 15%, no more than about 10%, nomore than about 8%, no more than about 6%, no more than about 5%, nomore than about 4%, no more than about 3% or less in at least onemeasured dimension. In some embodiments, disclosed herein is apopulation of nanoparticles, where at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, andideally about 100% of the particles are of the same size. Size deviationcan be measured as root mean square (“rms”) of the diameter, with thepopulation having less than about 30% rms, preferably less than about20% rms, more preferably less than about 10% rms. Size deviation can beless than about 10% rms, less than about 9% rms, less than about 8% rms,less than about 7% rms, less than about 6% rms, less than about 5% rms,less than about 3% rms, or ranges between any two of these values. Sucha collection of particles is sometimes referred to as being a“monodisperse” population.

The color (emitted light) of a nanoparticle can be “tuned” by varyingthe size and composition of the particle. Nanoparticles as disclosedherein can absorb a wide spectrum of wavelengths, and emit a relativelynarrow wavelength of light. The excitation and emission wavelengths aretypically different, and non-overlapping. The nanoparticles of amonodisperse population may be characterized in that they produce afluorescence emission having a relatively narrow wavelength band.Examples of emission widths include less than about 200 nm, less thanabout 175 nm, less than about 150 nm, less than about 125 nm, less thanabout 100 nm, less than about 75 nm, less than about 60 nm, less thanabout 50 nm, less than about 40 nm, less than about 30 nm, less thanabout 20 nm, and less than about 10 nm. In some embodiments, the widthof emission is less than about 60 nm full width at half maximum (FWHM),or less than about 50 nm FWHM, and sometimes less than about 40 nm FWHM,less than about 30 nm FWHM or less than about 20 nm FWHM. In someembodiments, the emitted light preferably has a symmetrical emission ofwavelengths.

The emission maxima of the disclosed nanoparticle and population thereofcan generally be at any wavelength from about 200 nm to about 2,000 nm.Examples of emission maxima include about 200 nm, about 400 nm, about600 nm, about 800 nm, about 1,000 nm, about 1,200 nm, about 1,400 nm,about 1,600 nm, about 1,800 nm, about 2,000 nm, and ranges between anytwo of these values.

As discussed previously, the disclosed nanoparticle or populationsthereof can comprise a core and a layered shell, wherein the shellincludes at least one inner (intermediate) shell layer comprising afirst shell material and at least one outer (external) shell layercomprising a second shell material, and wherein the layered shell issubstantially uniform in coverage around the core and is substantiallyfree of defects.

Thus, in one aspect, the nanoparticle or population thereof comprises acore (M¹Y) and a layered shell, wherein the shell comprises m innershell monolayers comprising a first shell material (M¹X)_(m) and n outershell monolayers comprising a second shell material (M²X), wherein M canbe a metal atom and X can be a non-metal atom, each of m and n isindependently an integer from 1 to 10, and the layered shell issubstantially uniform in coverage around the core and is substantiallyfree of defects. In specific embodiments, the sum of m+n is 3-20, or5-14, or 6-12, or 7-10.

In certain embodiments, the disclosed nanoparticles can further compriseone or more additional shell layers between the at least one inner shelllayer and the at least one outer shell layer.

In some embodiments, the nanoparticle core and population thereof canhave a first bandgap energy and the first shell material can have asecond bandgap energy, wherein the second bandgap energy can be greaterthan the first bandgap energy.

In a further aspect, provided herein is a nanoparticle or populationthereof comprising a core and a layered shell, wherein the shellcomprises sequential monolayers comprising an alloyed multi-componentshell material of the form M¹ _(x)M² _(y)X, where M¹ and M² can be metalatoms and X can be a non metal atom, where the composition becomessuccessively enriched in M² as the monolayers of shell material aredeposited, where x and y represent the ratio of M¹ and M² in the shellmaterial, and wherein the monolayered shell is substantially uniform incoverage around the core and is substantially free of defects. In someembodiments, the layered shell sometimes has about 3-20 monolayers ofshell material, sometimes about 5-14 monolayers of shell material,sometimes about 6-12 monolayers of shell material, or sometimes about7-10 monolayers of shell material.

In one aspect, provided herein is a nanoparticle or population thereofcomprising a core and a layered shell having a gradient potential,wherein the shell comprises at least one inner shell layer and at leastone outer shell layer, and wherein the layered shell is substantiallyuniform in coverage around the core and is substantially free ofdefects.

The layered shell may be engineered such that the sequential monolayersare selected to provide a gradient potential from the nanoparticle coreto the outer surface of the nanoparticle shell. The steepness of thepotential gradient may vary depending on the nature of the shellmaterials selected for each monolayer or group of monolayers. Forexample, a nanoparticle comprising several sequential monolayers of thesame shell material may reduce the potential through a series of steps,while a more continuous gradient may be achievable through the use ofsequential monolayers of a multi-component alloyed shell material. Insome embodiments, both single component and multi-component shellmaterials may be applied as different monolayers of a multi-layer shellon a nanoparticle.

The nanoparticles can be synthesized as disclosed to the desired size bysequential, controlled addition of materials to build and/or applymonolayers of shell material to the core. This is in contrast toconventional methods of adding shells where materials (e.g., diethylzincand bis(trimethylsilyl)sulfide) are added together. Sequential additionpermits the formation of thick (e.g., >2 nm) relatively uniformindividual shells (e.g., uniform size and depth) on a core. The layeradditions generally require the addition of an appropriate amount of theshell precursors to form a single monolayer, based on the starting sizeof the underlying core. This means that as each monolayer of shellmaterial is added, a new “core” size must be determined by taking theprevious “core” size and adding to it the thickness of just-added shellmonolayer. This leads to a slightly larger volume of the following shellmaterial needing to be added for each subsequent monolayer of shellmaterial being added.

Each monolayer of shell material can be independently selected, and maybe made up of a single component, or may comprise a multi-component(e.g., alloyed, etc.) shell material. In some embodiments, it issuitable to apply one or more sequential monolayers of a first shellmaterial, followed by one or more sequential monolayers of a secondshell material. This approach allows the deposition of at least oneinner shell layer of a material having a bandgap and lattice sizecompatible with the core, followed by the deposition of at least oneouter shell layer of a material having a bandgap and lattice sizecompatible with the inner shell layer. In some embodiments, multiplesequential monolayers of a single shell material can be applied toprovide a uniform shell of a desired number of monolayers of a singleshell material; in these embodiments, the first and second shellmaterials are the same. In other embodiments, sequential monolayers ofan alloyed shell material are applied, where the ratio of the componentsvaries such that the composition becomes successively enriched in onecomponent of the multi-component mixture as the successive monolayers ofshell material are deposited.

In some embodiments, the layered shell can be about 3-20 monolayers ofshell material thick, sometimes about 5-14 monolayers of shell materialthick, sometimes about 6-12 monolayers of shell material thick orsometimes about 7-10 monolayers of shell material thick. In someembodiments, at least one inner shell layer can be comprised of about3-5 monolayers, sometimes about 3-7 monolayers, of the first shellmaterial. In other embodiments, at least one outer shell layer can becomprised of about 3-5 monolayers, sometimes about 3-7 monolayers, ofthe second shell material. In some embodiments, the inner shell layercan be at least 3 monolayers thick; in other embodiments, the outershell layer can be at least 3 monolayers thick. The individualmonolayers can be formed by the controlled, sequential addition of thelayer materials methods described herein. The monolayers may not alwaysbe completely distinct as they may, in some embodiments, be a latticingbetween the surfaces of contacting monolayers.

In certain embodiments, provided herein are nanoparticles having athick, uniform, layered shell, as described herein, wherein the corecomprises CdSe, the at least one inner shell layer comprises CdS, andthe at least one outer shell layer comprises ZnS. In a particularembodiment, provided herein is a nanoparticle or population thereofhaving a CdSe core and a layered shell comprising 4CdS+3.5ZnS layers. Insome embodiments, provided herein is a nanoparticle which consistsessentially of CdSe/4CdS−3.5ZnS.

Also disclosed herein are methods of making a nanoparticle andpopulation thereof with modulated, reduced or no fluorescenceintermittency or “blinking”. These nanoparticles can be small,photostable, bright, highly FRET efficient or some combination thereof.These nanoparticles can have a multi-shell layered core achieved by asequential shell material deposition process, whereby one shell materialis added at a time, to provide a nanoparticle having a substantiallyuniform shell of desired thickness which is substantially free ofdefects.

In one aspect, provided herein is a method for making a nanoparticle orpopulation thereof with modulated, reduced or no fluorescenceintermittency, comprising: providing a mixture comprising a core and atleast one coordinating solvent; adding a first inner shell precursoralternately with a second inner shell precursor in layer additions, toform an inner shell layer which is a desired number of layers thick; andadding a first outer shell precursor alternately with a second outershell precursor in layer additions, to form an outer shell layer whichis a desired number of layers thick. If the coordinating solvent of isnot amine, the method further comprises an amine in.

In some embodiments, the mixture can be heated to a temperature which issuitable for shell formation before and/or after every sequentialaddition of a shell precursor. In some embodiments, the shell issubstantially uniform in coverage around the core and is substantiallyfree of defects. In some embodiments, the resulting nanoparticles have adiameter of less than about 15 nm. In other embodiments, thenanoparticles have a diameter of between about 6 nm to about 10 nm. Thenanoparticles made by this method can have quantum yields greater thanabout 80%. The nanoparticle made by this method can have on-timefractions (i.e., ratio of the time which nanoparticle emission is turned“on” when the nanoparticle is excited) of greater than about 0.80 (undermoderate to high excitation energy).

In another aspect, provided herein is a method for making a FRET capablenanoparticle and populations thereof with modulated, reduced or nofluorescence intermittency, comprising: (a) providing a mixturecomprising a plurality of nanocrystal cores and at least onecoordinating solvent; (b) adding a first intermediate shell precursoralternately with a second intermediate shell precursor in layeradditions to form an intermediate shell layer on each of the pluralityof nanocrystal cores, wherein the intermediate shell layer is comprisedof more than one monolayer; (c) adding a first external shell precursoralternately with a second external shell precursor in layer additions toform an external shell layer on each of the plurality of nanocrystalcores, wherein the external shell layer is disposed on top of theintermediate shell layer and is comprised of more than one monolayer;(d) adding an aqueous solution comprising a hydrophilic ligand; and (e)maintaining the mixture under conditions which cause the plurality ofnanocrystals to migrate into an aqueous phase. If the coordinatingsolvent is not an amine, at least one amine can be included in step (a).In some embodiments, the resulting population of FRET capablenon-blinking nanoparticles has a α_(on) value which is less than about1.4. In other embodiments, the resulting population of FRET capablenon-blinking nanoparticles has an on-time fraction of least about 0.8(under moderate to high excitation energy). In some embodiments, theresulting population of FRET capable non-blinking nanoparticles hasdiameters which are less than about 15 nm. In some embodiments, theresulting population of FRET capable non-blinking nanoparticles has aFRET efficiency of at least 20%. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a quantumyield of at least about 40%.

In some embodiments, the methods disclosed above utilize a one step or atwo step ligand exchange process to replace the hydrophobic ligands onthe nanoparticles with hydrophilic ligands to cause the plurality ofnanocrystals to migrate into the aqueous phase. See PCT ApplicationSerial No. PCT/US09/53018 and PCT/US09/59456 which are expresslyincorporated herein by reference as if set forth in full.

In another aspect, provided herein is a method for making a FRET capablenanoparticle and populations thereof with modulated, reduced or nofluorescence intermittency, comprising: providing a mixture comprising aplurality of nanocrystal cores, functionalized organophosphorous-basedhydrophilic ligands and at least one coordinating solvent; adding afirst intermediate shell precursor alternately with a secondintermediate shell precursor in layer additions to form an intermediateshell layer on each of the plurality of nanocrystal cores; and adding afirst external shell precursor alternately with a second external shellprecursor in layer additions to form an external shell layer on each ofthe plurality of nanocrystal cores. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has an α_(on)value which is less than about 1.4. In other embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has an on-timefraction of least about 0.8. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has diameterswhich are less than about 15 nm. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a FRETefficiency of at least 20%. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a quantumyield of at least about 40%.

In some embodiments, the functionalized organophosphorous-basedhydrophilic ligands are multi-functional surface ligands which include aphosphonate/phosphinate nanocrystal binding center, a linker, and afunctional group, which imparts functionality on the nanocrystal. Asused herein the term “functional group” may refer to a group whichaffects reactivity, solubility, or both reactivity and solubility whenpresent on a multi-functional surface ligand. Embodiments can include awide variety of functional groups which can impart various types offunctionality on the nanocrystal including hydrophilicity,water-solubility, or dispersibility and/or reactivity, and thefunctionality may generally not include only hydrophobicity or onlysolubility in organic solvents without increasing reactivity. Forexample, a functional group which is generally hydrophobic but whichincreases reactivity such as an alkene or alkyne and certain esters andethers can be encompassed by embodiments, whereas alkyl groups, which donot generally impart reactivity but increase hydrophobicity may beexcluded.

In certain embodiments, the FRET capable and non-blinking nanoparticlesproduced by the disclosed methods may be coated with ligands whichimpart water solubility and/or reactivity on the nanoparticle obviatingthe need for ligand replacement. Without wishing to be bound by theory,eliminating ligand replacement may provide more consistent thermodynamicproperties, which may lead to reduction in variability of coating andless loss of quantum yield, among other improvements in the propertiesof nanoparticles produced by the methods embodied herein. Eliminatingligand replacement may also allow for the production of nanoparticleshaving a wide variety of functional groups associated with the coating.In particular, while ligand replacement is generally limited toproduction of nanoparticles having amine and/or carboxylic acidfunctional groups, in various embodiments, the skilled artisan maychoose among numerous functional groups when preparing themulti-functional ligands and may, therefore, generate nanoparticleswhich provide improved water-solubility or water-dispersity and/orsupport improved crosslinking and/or improved reactivity with cargomolecules. See PCT Application Serial No. PCT/US09/59117 which isexpressly incorporated herein by reference as if set forth in full.

In another aspect, provided herein is a method of making a nanoparticleor population thereof comprising a core and a layered gradient shell,wherein the shell comprises an multi-component (e.g., alloy, etc.) shellmaterial of the form M¹ _(x)M² _(y)X, where x and y represent the ratioof M¹ and M² in the shell material. The method comprising: (a) providinga mixture comprising a core, at least one coordinating solvent; (b)heating said mixture to a temperature suitable for formation of theshell layer; and (c) adding a first inner shell precursor comprising M¹_(x) and M² _(y) alternately with a second inner shell precursorcomprising X in layer additions, wherein the ratio of y to x graduallyincreases in sequential layer additions, such that the shell layersbecomes successively enriched in M², to form a layered gradient shellwhich is a desired number of monolayers thick. If the coordinatingsolvent is not an amine, at least one amine can be included in step (a).

In one embodiment, the method described above provides a nanoparticlehaving a layered gradient shell, wherein the core comprises CdSe and theshell comprises sequential layers of Cd_(x)Zn_(y)S, where the ratio of yto x increases gradually from the innermost shell layer to the outermostshell layer, to provide a layered gradient shell with a finely gradedpotential. In some such embodiments, the outermost shell layer isessentially pure ZnS. In some embodiments, the percent of Zn in thegradient shell varies from less than about 10% at the innermost shelllayer to greater than about 80% at the outermost shell layer.

Typically, the heating steps in the disclosed methods are conducted at atemperature within the range of about 150-350° C., more preferablywithin the range of about 200-300° C. In some embodiments, thetemperature suitable for formation of at least one inner shell layer isabout 215° C. In some embodiments, the temperature suitable forformation of at least one outer shell layer is about 245° C. It isunderstood that the above ranges are merely exemplary and are notintended to be limiting in any manner as the actual temperature rangesmay vary, dependent upon the relative stability of the precursors,ligands, and solvents. Higher or lower temperatures may be appropriatefor a particular reaction. The determination of suitable time andtemperature conditions for providing nanoparticles is within the levelof skill in the art using routine experimentation.

It can be advantageous to conduct the nanoparticle-forming reactionsdescribed herein with the exclusion of oxygen and moisture. In someembodiments the reactions are conducted in an inert atmosphere, such asin a dry box. The solvents and reagents are also typically rigorouslypurified to remove moisture and oxygen and other impurities, and aregenerally handled and transferred using methods and apparatus designedto minimize exposure to moisture and/or oxygen. In addition, the mixingand heating steps can be conducted in a vessel which is evacuated andfilled and/or flushed with an inert gas such as nitrogen. The fillingcan be periodic or the filling can occur, followed by continuousflushing for a set period of time.

In some embodiments, the at least one coordinating solvent comprises atrialkylphosphine, a trialkylphosphine oxide, a phosphonic acid, or amixture of these. Sometimes, the at least one coordinating solventcomprises TOP, TOPO, TDPA, OPA or a mixture of these. The solvent forthese reactions often comprises a primary or secondary amine, forexample, decylamine, hexadecylamine, or dioctylamine. In someembodiments, the amine is decylamine. In some embodiments, the firstinner shell precursor is Cd(OAc)₂ and the second inner shell precursoris bis(trimethylsilyl)sulfide (TMS₂S). Sometimes, the first and secondinner shell precursors are added as a solution in TOP. In someembodiments, the first outer shell precursor is Et₂Zn and the secondinner shell precursor is TMS₂S. Sometimes, the first and second outershell precursors are added as a solution in TOP.

In certain embodiments, the disclosed nanoparticles may be preparedusing the method described herein to build a layered CdS—ZnS shell on aCdSe quantum size core. The shells for these materials can have varyingnumbers of layers of CdS and ZnS. Prototypical materials containing aCdSe core and approximately 4 monolayers CdS and 3.5 monolayers of ZnS(the final 0.5 monolayer is essentially pure Zn), or a CdSe core and 9monolayers CdS and 3.5 monolayers of ZnS were prepared as described inthe examples.

In some embodiments, for either the inner or outer layer, or both, lessthan a full layer of the appropriate first shell precursor can be addedalternately with less than a full layer of the appropriate second shellprecursor, so the total amount of the first and second shell precursorrequired is added in two or more portions. Sometimes, the portion isabout 0.25 monolayers of shell material, so that the 4 portions of 0.25monolayer of first shell precursor are added alternately with 4 portionsof 0.25 monolayer of second shell precursor; sometimes the portion isabout 0.5 monolayers of shell material, and sometimes about 0.75monolayers of shell material.

Examples of compounds useful as the first precursor can include, but arenot limited to: organometallic compounds such as alkyl metal species,salts such as metal halides, metal acetates, metal carboxylates, metalphosphonates, metal phosphinates, metal oxides, or other salts. In someembodiments, the first precursor provides a neutral species in solution.For example, alkyl metal species such as diethylzinc (Et₂Zn) or dimethylcadmium are typically considered to be a source of neutral zinc atoms(Zn⁰) in solution. In other embodiments, the first precursor provides anionic species (i.e., a metal cation) in solution. For example, zincchloride (ZnCl₂) and other zinc halides, zinc acetate (Zn(OAc)₂) andzinc carboxylates are typically considered to be sources of Zn²⁺ cationsin solution.

By way of example only, suitable first precursors providing neutralmetal species include dialkyl metal sources, such as dimethyl cadmium(Me₂Cd), diethyl zinc (Et₂Zn), and the like. Suitable first precursorsproviding metal cations in solution include, e.g., cadmium salts, suchas cadmium acetate (Cd(OAc)₂), cadmium nitrate (Cd(NO₃)₂), cadmium oxide(CdO), and other cadmium salts; and zinc salts such as zinc chloride(ZnCl₂), zinc acetate (Zn(OAc)₂), zinc oleate (Zn(oleate)₂), zincchloro(oleate), zinc undecylenate, zinc salicylate, and other zincsalts. In some embodiments, the first precursor is salt of Cd or Zn. Insome embodiments, it is a halide, acetate, carboxylate, or oxide salt ofCd or Zn. In other embodiments, the first precursor is a salt of theform M(O₂CR)X, wherein M is Cd or Zn; X is a halide or O₂CR; and R is aC4-C24 alkyl group which is optionally unsaturated. Other suitable formsof Groups 2, 12, 13 and 14 elements useful as first precursors are knownin the art.

Precursors useful as the “second” precursor in the disclosed methodsinclude compounds containing elements from Group 16 of the PeriodicTable of the Elements (e.g., S, Se, Te, and the like), compoundscontaining elements from Group 15 of the Periodic Table of the Elements(N, P, As, Sb, and the like), and compounds containing elements fromGroup 14 of the Periodic Table of the Elements (Ge, Si, and the like).Many forms of the precursors can be used in the disclosed methods. Itwill be understood that in some embodiments, the second precursor willprovide a neutral species in solution, while in other embodiments thesecond precursor will provide an ionic species in solution.

When the first precursor comprises a metal cation, the second precursorcan provide an uncharged (i.e., neutral) non-metal atom in solution. Infrequent embodiments, when the first precursor comprises a metal cation,the second precursor contributes a neutral chalcogen atom, most commonlyS⁰, Se⁰ or Te⁰.

Suitable second precursors for providing a neutral chalcogen atominclude, for example, elemental sulfur (often as a solution in an amine,e.g., decylamine, oleylamine, or dioctylamine, or an alkene, such asoctadecene), and tri-alkylphosphine adducts of S, Se and Te. Suchtrialkylphosphine adducts are sometimes described herein as R3P═X,wherein X is S, Se or Te, and each R is independently H, or a C1-C24hydrocarbon group which can be straight-chain, branched, cyclic, or acombination of these, and which can be unsaturated. Exemplary secondprecursors of this type include tri-n (butylphosphine)selenide (TBP=Se),tri-n-(octylphosphine)selenide (TOP=Se), and the corresponding sulfurand tellurium reagents, TBP=S, TOP=S, TBP=Te and TOP=Te. These reagentsare frequently formed by combining a desired element, such as Se, S, orTe with an appropriate coordinating solvent, e.g., TOP or TBP.Precursors which provide anionic species under the reaction conditionsare typically used with a first precursor which provides a neutral metalatom, such as alkylmetal compounds and others described above or knownin the art.

In some embodiments, the second precursor provides a negatively chargednon-metal ion in solution (e.g., S-2, Se-2 or Te-2). Examples ofsuitable second precursors providing an ionic species include silylcompounds such as bis(trimethylsilyl)selenide ((TMS)₂Se),bis(trimethylsilyl)sulfide ((TMS)₂S) and bis(trimethylsilyl)telluride((TMS)₂Te). Also included are hydrogenated compounds such as H2Se, H2S,H2Te; and metal salts such as NaHSe, NaSH or NaHTe. In this situation,an oxidant can be used to oxidize a neutral metal species to a cationicspecies which can react with the anionic precursor in a ‘matched’reaction, or an oxidant can be used increase the oxidation state of theanionic precursor to provide a neutral species which can undergo a‘matched’ reaction with a neutral metal species.

Other exemplary organic precursors are described in U.S. Pat. Nos.6,207,229 and 6,322,901 to Bawendi et al., and synthesis methods usingweak acids as precursor materials are disclosed by Qu et al., (2001),Nano Lett., 1(6):333-337, the disclosures of each of which areincorporated herein by reference in their entirety.

Both the first and the second precursors can be combined with anappropriate solvent to form a solution for use in the disclosed methods.The solvent or solvent mixture used to form a first precursor solutionmay be the same or different from that used to form a second precursorsolution. Typical coordinating solvents include alkyl phosphines, alkylphosphine oxides, alkyl phosphonic acids, alkyl phosphinic acids, orcarboxylic acid containing solvents, or mixtures of these.

Suitable reaction solvents include, by way of illustration and notlimitation, hydrocarbons, amines, alkyl phosphines, alkyl phosphineoxides, carboxylic acids, ethers, furans, phosphoacids, pyridines andmixtures thereof. The solvent may actually comprise a mixture ofsolvents, often referred to in the art as a “solvent system”. In someembodiments, the solvent comprises at least one coordinating solvent. Insome embodiments, the solvent system comprises a secondary amine and atrialkyl phosphine (e.g., TBP or TOP) or a trialkylphosphine oxide(e.g., TOPO). If the coordinating solvent is not an amine, an amine canbe included.

A coordinating solvent might be a mixture of an essentiallynon-coordinating solvent such as an alkane and a ligand as definedbelow.

Suitable hydrocarbons include alkanes, alkenes and aromatic hydrocarbonsfrom 10 to about 30 carbon atoms; examples include octadecene andsqualane. The hydrocarbon may comprise a mixture of alkane, alkene andaromatic moieties, such as alkylbenzenes (e.g., mesitylene).

Suitable amines include, but are not limited to, monoalkylamines,dialkylamines, and trialkylamines, for example dioctylamine, oleylamine,decylamine, dodecylamine, hexyldecylamine, and so forth. Alkyl groupsfor these amines typically contain about 6-24 carbon atoms per alkyl,and can include an unsaturated carbon-carbon bond, and each aminetypically has a total number of carbon atoms in all of its alkyl groupscombined of about 10-30 carbon atoms.

Exemplary alkyl phosphines include, but are not limited to, the trialkylphosphines, tri-n-butylphosphine (TBP), tri-n-octylphosphine (TOP), andso forth. Alkyl groups for these phosphines contain about 6-24 carbonatoms per alkyl, and can contain an unsaturated carbon-carbon bond, andeach phosphine has a total number of carbon atoms in all of its alkylgroups combined of about 10-30 carbon atoms.

Suitable alkyl phosphine oxides include, but are not limited to, thetrialkyl phosphine oxide, tri-n-octylphosphine oxide (TOPO), and soforth. Alkyl groups for these phosphine oxides contain about 6-24 carbonatoms per alkyl, and can contain an unsaturated carbon-carbon bond, andeach phosphine oxide has a total number of carbon atoms in all of itsalkyl groups combined of about 10-30 carbon atoms.

Exemplary fatty acids include, but are not limited to, stearic, oleic,palmitic, myristic and lauric acids, as well as other carboxylic acidsof the formula R—COOH, wherein R is a C6-C24 hydrocarbon group and cancontain an unsaturated carbon-carbon bond. It will be appreciated thatthe rate of nanocrystal growth generally increases as the length of thefatty acid chain decreases.

Exemplary ethers and furans include, but are not limited to,tetrahydrofuran and its methylated forms, glymes, and so forth.

Suitable phosphonic and phosphinic acids include, but are not limited tohexylphosphonic acid (HPA), tetradecylphosphonic acid (TDPA), andoctylphosphinic acid (OPA), and are frequently used in combination withan alkyl phosphine oxide such as TOPO. Suitable phosphonic andphosphinic acids are of the formula RPO₃H₂ or R₂PO₂H, wherein each R isindependently a C6-C24 hydrocarbon group and can contain an unsaturatedcarbon-carbon bond.

Exemplary pyridines include, but are not limited to, pyridine, alkylatedpyridines, nicotinic acid, and so forth.

Suitable alkenes include, e.g., octadecene and other C4-C24 hydrocarbonswhich are unsaturated.

Nanoparticle core or shell precursors can be represented as a M-sourceand an X-donor. The M-source can be an M-containing salt, such as ahalide, carboxylate, phosphonate, carbonate, hydroxide, or diketonate,or a mixed salt thereof (e.g., a halo carboxylate salt, such asCd(halo)(oleate)), of a metal, M, in which M can be, e.g., Cd, Zn, Mg,Hg, Al, Ga, In, or Tl. In the X-donor, X can be, e.g., O, S, Se, Te, N,P, As, or Sb. The mixture can include an amine, such as a primary amine(e.g., a C8-C20 alkyl amine). The X donor can include, for example, aphosphine chalcogenide, a bis(trialkylsilyl)chalcogenide, a dioxygenspecies, an ammonium salt, or a tris(trialkylsilyl)phosphine, or thelike.

The M-source and the X donor can be combined by contacting a metal, M,or an M-containing salt, and a reducing agent to form an M-containingprecursor. The reducing agent can include an alkyl phosphine, a 1,2-diolor an aldehyde, such as a C₆-C₂₀ alkyl diol or a C₆-C₂₀ aldehyde.

Suitable M-containing salts include, for example, cadmiumacetylacetonate, cadmium iodide, cadmium bromide, cadmium chloride,cadmium hydroxide, cadmium carbonate, cadmium acetate, cadmium oxide,zinc acetylacetonate, zinc iodide, zinc bromide, zinc chloride, zinchydroxide, zinc carbonate, zinc acetate, zinc oxide, magnesiumacetylacetonate, magnesium iodide, magnesium bromide, magnesiumchloride, magnesium hydroxide, magnesium carbonate, magnesium acetate,magnesium oxide, mercury acetylacetonate, mercury iodide, mercurybromide, mercury chloride, mercury hydroxide, mercury carbonate, mercuryacetate, aluminum acetylacetonate, aluminum iodide, aluminum bromide,aluminum chloride, aluminum hydroxide, aluminum carbonate, aluminumacetate, gallium acetylacetonate, gallium iodide, gallium bromide,gallium chloride, gallium hydroxide, gallium carbonate, gallium acetate,indium acetylacetonate, indium iodide, indium bromide, indium chloride,indium hydroxide, indium carbonate, indium acetate, thalliumacetylacetonate, thallium iodide, thallium bromide, thallium chloride,thallium hydroxide, thallium carbonate, or thallium acetate. SuitableM-containing salts also include, for example, carboxylate salts, such asoleate, stearate, myristate, and palmitate salts, mixed halo carboxylatesalts, such as M(halo)(oleate) salts, as well as phosphonate salts.

Alkyl is a branched or unbranched saturated hydrocarbon group of 1 to100 carbon atoms, preferably 1 to 30 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well ascycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Optionally, an alkyl can contain 1 to 6 linkages selected from the groupconsisting of —O—, —S—, -M- and —NR— where R is hydrogen, or C1-C8 alkylor lower alkenyl.

The X donor is a compound capable of reacting with the M-containing saltto form a material with the general formula MX. The X donor is generallya chalcogenide donor or a phosphine donor, such as a phosphinechalcogenide, a bis(silyl) chalcogenide, dioxygen, an ammonium salt, ora tris(trialkylsilyl) phosphine. Suitable X donors include dioxygen,elemental sulfur, bis(trimethylsilyl) selenide ((TMS)₂Se), trialkylphosphine selenides such as (tri-n-octylphosphine) selenide (TOPSe) or(tri-n-butylphosphine) selenide (TBPSe), trialkyl phosphine telluridessuch as (tri-n-octylphosphine) telluride (TOPTe) orhexapropylphosphorustriamide telluride (HPPTTe),bis(trimethylsilyl)telluride ((TMS)₂Te), sulfur,bis(trimethylsilyl)sulfide ((TMS)₂S), a trialkyl phosphine sulfide suchas (tri-n-octylphosphine) sulfide (TOPS), tris(dimethylamino) arsine, anammonium salt such as an ammonium halide (e.g., NH₄Cl),tris(trimethylsilyl) phosphide ((TMS)₃P), tris(trimethylsilyl) arsenide((TMS)₃As), or tris(trimethylsilyl) antimonide ((TMS)₃Sb). In certainembodiments, the M donor and the X donor can be moieties within the samemolecule.

Ligand Exchange Processes for Coating Nanoparticles

Provided herein are ligand exchange processes that permit efficientconversion of a conventional hydrophobic nanoparticle or populationthereof into a water-dispersible and functionalized nanoparticle orpopulation of nanoparticles. It also permits preparation of smallnanoparticles which are highly stable and bright enough to be useful inbiochemical and biological assays. The resulting nanoparticles can alsobe linked to a target molecule or cell or enzyme (e.g., polymerase) ofinterest.

Typically, the nanoparticle used for this process is a core/shellnanocrystal which is coated with a hydrophobic ligand such astetradecylphosphonic acid (TDPA), trioctylphosphine oxide (TOPO),trioctyl phosphine (TOP), octylphosphonic acid (OPA), and the like, or amixture of such ligands; these hydrophobic ligands typically have atleast one long-chain alkyl group, i.e. an alkyl group having at least 8carbons, or for the phosphine/phosphine oxide ligands, this hydrophobiccharacter may be provided by two or three alkyl chains on a singleligand molecule having a total of at least 10 carbon atoms. Therefore,in some embodiments, the surface of the core/shell nanocrystal orpopulation thereof can be coated with varying quantities of TDPAhydrophobic ligands prior to replacement with hydrophilic ligand(s). Forexample, TDPA can represent at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about80%, at least about 95%, at least about 98%, at least about 99% or moreof the total surface ligands coating the core/shell nanoparticles.Moreover, certain hydrophobic ligands show an unexpected and apparentease of replacement with the hydrophilic ligand. For example,nanoparticles with OPA on the surface have been observed to transferinto aqueous buffer more readily and more completely than the same typeof core-shell with TDPA on the surface. Therefore, in some embodiments,the surface of the core/shell nanocrystal or populations thereof can becoated with varying quantities of OPA hydrophobic ligands prior toreplacement with hydrophilic ligand(s). For example, OPA can representat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 80%, at least about 95%,at least about 98%, at least about 99% or more of the total surfaceligands coating the core/shell nanocrystal.

In one aspect, provided herein is a “one-step” ligand exchange processto apply various types of ligands to the surface of a nanoparticle, bysubstituting a desired hydrophilic ligand for a conventional hydrophobicligand like TOPO, TOP, TDPA, OPA, and the like. The process steps,comprising: providing a nanocrystal coated with a surface layercomprising a hydrophobic ligand, and dissolved or dispersed in anon-aqueous solvent, contacting the nanocrystal dispersion with a phasetransfer agent and an aqueous solution comprising a hydrophilic ligand,to form a biphasic mixture having an aqueous phase and a non-aqueousphase and maintaining the mixture under conditions that cause thenanocrystal to migrate from the non-aqueous solvent into the aqueousphase. See PCT Application Serial No. PCT/US09/53018 which is expresslyincorporated herein by reference as if set forth in full.

The ‘one-step’ ligand exchange process described herein utilizes phasetransfer catalysts which are particularly effective, and provide fasterexchange reactions. Butanol has been utilized as a phase transfercatalyst for this type of exchange reaction; however, the reaction takesseveral days typically, and requires heating to about 70° C. The timefor this reaction exposes the nanoparticles to these reaction conditionsfor a long period of time, which may contribute to some reduction in itsultimate stability. The embodiments disclosed herein provide moreefficient conditions which achieve ligand exchange more rapidly, thusbetter protecting the nanoparticles. As a result of accelerating theexchange reaction and allowing use of milder conditions, these phasetransfer catalysts produce higher quality nanoparticles.

The phase transfer agent for this process can be a crown ether, a PEG, atrialkylsulfonium, a tetralkylphosphonium, and an alkylammonium salt, ora mixture of these. In some embodiments, the phase transfer agent is18-crown-6, 15-crown-5, or 12-crown-4. In some embodiments, the phasetransfer agent is a PEG, which can have a molecular weight from about500 to about 5000. In some embodiments, the phase transfer agent is atrialkylsulfonium, tetralkylphosphonium, or alkylammonium (includingmonoalkylammonium, dialkylammonium, trialkylammonium andtetralkylammonium) salt.

Tetralkylammonium salts are sometimes preferred as phase transferagents. Examples of suitable tetralkylammonium salts includetriethylbenzyl ammonium, tetrabutylammonium, tetraoctylammonium, andother such quaternary salts. Other tetralkylammonium salts, where eachalkyl group is a C1-C12 alkyl or arylalkyl group, can also be used.Typically, counting all of the carbons on the alkyl groups of atrialkylsulfonium, tetralkylphosphonium, and alkylammonium salt, thephase transfer agent will contain a total of at least 2 carbons, atleast 10 carbons and preferably at least 12 carbon atoms. Each of thetrialkylsulfonium, tetralkylphosphonium, and alkylammonium salts has acounterion associated with it; suitable counterions include halides,preferably chloride or fluoride; sulfate, nitrate, perchlorate, andsulfonates such as mesylate, tosylate, or triflate; mixtures of suchcounterions can also be used. The counterion can also be a buffer orbase, such as borate, hydroxide or carbonate; thus, for example,tetrabutylammonium hydroxide can be used to provide the phase transfercatalyst and a base. Specific phase transfer salts for use in thesemethods include tetrabutylammonium chloride (or bromide) andtetraoctylammonium bromide (or chloride).

Suitable hydrophilic ligands are organic molecules which provide atleast one binding group to associate tightly with the surface of ananocrystal. The hydrophilic ligand typically is an organic moietyhaving a molecular weight between about 100 and 1500, and containsenough polar functional groups to be water soluble. Some examples ofsuitable hydrophilic ligands include small peptide having 2-10 aminoacid residues (preferably including at least one histidine or cysteineresidue), mono- or polydentate thiol containing compounds.

Following ligand exchange, the surface layer can optionally becrosslinked.

In another aspect, provided herein is a “two-step” ligand exchangeprocess to apply various types of ligands to the surface of ananoparticle, by substituting a desired hydrophilic ligand for aconventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and the like.The process involves the removal of phosphonate or phosphinate ligandsfrom the surface of a nanoparticle or nanocrystal by treatment withsulfonate reagents, particularly silylsulfonate derivatives of weakbases or other poorly coordinating groups.

The process steps, comprising: providing a nanocrystal whose surfacecomprises a phosphonate ligand, contacting the nanocrystal with asulfonate reagent in an organic solvent, contacting the sulfonate ligandcoated nanocrystal with a functionalized organic molecule (i.e.,hydrophilic ligand) comprising at least one nanocrystal surfaceattachment group, contacting the nanocrystal dispersion with an aqueoussolution to form a biphasic mixture having an aqueous phase and anon-aqueous phase, and maintaining the biphasic mixture under conditionswhich cause the nanocrystal to migrate from the non-aqueous phase intothe aqueous phase. See PCT Application Serial No. PCT/US09/59456 whichis expressly incorporated herein by reference as if set forth in full.

The result of this removal of phosphonate ligands is replacement of thephosphonates with the weakly coordinating groups. One example is the useof silyl sulfonates, such as trimethylsilyl triflate, to form asulfonate-coated nanoparticle. Triflate is a conventional/common namefor a trifluoromethanesulfonyloxy group, CF₃SO₂O—.

The same type of replacement process can also occur on nanoparticleshaving phosphinic acid ligands of the formula R₂P(═O)—OH or onnanoparticles having carboxylic acid ligands of the formula RC(═O)—OH,which could be incorporated on the surface of a nanocrystal by knownmethods; R can be a C₁-C₂₄ hydrocarbon group in these phosphinates, andthe two R groups can be the same or different. Thus, it is understoodthat when phosphonate-containing nanocrystals are described herein,phosphinate-containing nanocrystals can be used instead, with similarresults.

This process provides a mild and selective method for removingphosphonate, phosphinate, and carboxylate ligands from the surface of ananocrystal. As a result, it provides a way for a user to remove thesegroups and replace them, without removing other ligands which are notdisplaced or affected by the silylsulfonate.

The sulfonate ligands can comprise an alkyl or aryl moiety linked to—SO₃X, where X can represent whatever the sulfonate group is attachedto. For example, where the sulfonate ligand is a sulfonate anion (i.e.,triflate), X would represent a nanocrystal, or the surface of ananocrystal. Some of the sulfonate embodiments disclosed herein can alsobe described with reference to feature ‘A’ of Formula I, as set forthbelow.

wherein R¹, R², R³ and A are each, independently, C1-C10 alkyl or C5-C10aryl; and each alkyl and aryl is optionally substituted.

The alkyl groups for Formula I compounds are independently selected, andcan be straight chain, branched, cyclic, or combinations of these, andoptionally can include a C1-C4 alkoxy group as a substituent. Typically,the alkyl groups are lower alkyls, e.g., C1-C4 alkyl groups which arelinear or branched. Methyl is one suitable example.

The aryl group for the compounds of Formula I can be phenyl, naphthyl ora heteroaryl having up to 10 ring members, and can be monocyclic orbicyclic, and optionally contain up to two heteroatoms selected from N,O and S as ring members in each ring. (It will be understood by thoseskilled in the art that the 5-membered aryl is a heteroaryl ring.)Phenyl is a preferred aryl group; and an aryl group is typically onlypresent if the other organic groups on the silicon other than thesulfonate are lower alkyls, and preferably they are each Me.

Examples of silylsulfonate ligands can include, but are not limited to:(trimethylsilyl)triflate, (triethylsilyl)triflate,(t-butyldimethylsilyl)triflate, (phenyldimethylsily)triflate,trimethylsilyl fluoromethanesulfonate, trimethylsilyl methanesulfonate,trimethylsilyl nitrophenylsulfonate, trimethylsilyltrifluoroethylsulfonate, trimethylsilyl phenylsulfonate, trimethylsilyltoluenesulfonate, diisopropylsilyl bis(trifluoromethanesulfonate),tertbutyldimethylsilyl trifluoromethanesulfonate, triisopropylsilyltrifluoromethanesulfonate and trimethylsilyl chlorosulfonate.

Examples of other sulfonate ligands can include, but are not limited to:trifluoromethanesulfonate (triflate), fluoromethanesulfonate,methanesulfonate (mesylate), nitrophenylsulfonate (nosylate),trifluorethylsulfonate, phenylsulfonate (besylate) and toluenesulfonate(tosylate).

Some suitable examples of the hydrophilic ligand are disclosed, forexample, in Naasani, U.S. Pat. Nos. 6,955,855; 7,198,847; 7,205,048;7,214,428; and 7,368,086. Suitable hydrophilic ligands also includeimidazole containing compounds such as peptides, particularlydipeptides, having at least one histidine residue, and peptides,particularly dipeptides, having at least one cysteine residue. Specificligands of interest for this purpose can include carnosine (whichcontains beta-alanine and histidine); His-Leu; Gly-His; His-Lys;His-Glu; His-Ala; His-His; His-Cys; Cys-His; His-Ile; His-Val; and otherdipeptides where His or Cys is paired with any of the common alpha-aminoacids; and tripeptides, such as Gly-His-Gly, His-Gly-His, and the like.The chiral centers in these amino acids can be the naturalL-configuration, or they can be of the D-configuration or a mixture of Land D. Thus a dipeptide having two chiral centers such as His-Leu can beof the L,L-configuration, or it can be L,D- or D,L; or it can be amixture of diastereomers.

Furthermore, suitable hydrophilic ligands can also include mono- orpolydentate thiol containing compounds, for example: monodentate thiolssuch as mercaptoacetic acid, bidentate thiols such as dihydrolipoic acid(DHLA), tridentate thiols such as compounds of Formula II-VII as shownbelow, and the like.

In compounds of Formula II-VII, R¹, R², R³ can independently be H, halo,hydroxyl, (—(C═O)—C₁-C₂₂, —(C═O)CF₃) alkanoyl, C₁-C₂₂ alkyl, C₁-C₂₂heteroalkyl, ((CO)OC₁-C₂₂) alkylcarbonato, alkylthio (C₁-C₂₂) or(—(CO)NH(C₁-C₂₀) or —(CO)N(C₁-C₂₀)₂) alkylcarbamoyl. In someembodiments, R¹, R², and R³ are different. In other embodiments, R¹, R²,and R³ are the same.

In compounds of Formula II-VII, IV, and R⁵ can independently be H,C₁-C₂₀ alkyl, C₆-C₁₈ aryl, C₁-C₂₂ heteroalkyl or C₁-C₂₂ heteroaryl. Insome embodiments, R⁴ and R⁵ are different. In other embodiments, R⁴ andR⁵ are the same.

In compounds of Formula II-VII, R⁶ can be H or a polyethylene glycolbased moiety of Formula VIII:

In certain embodiments of Formula VIII, R⁷ can be —NH₂, —N₃, —NHBoc,—NHFmoc, —NHCbz, —COOH, —COOt-Bu, —COOMe, iodoaryl, hydroxyl, alkyne,boronic acid, allylic alcohol carbonate, —NHBiotin, —(CO)NHNHBoc,—(CO)NHNHFmoc or —OMe. In some embodiments, n can be an integer from 1to 100.

In still further embodiments, the tridentate thiol ligands can be acompound of Formula IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII,XIX, XX, XXI, XXII, XXIII or XXIV:

Functionalized TDPA Ligands on Nanoparticles

Provided herein are methods for preparing water-soluble semi-conducting,insulating, or metallic nanoparticles including the steps of admixingone or more nanocrystal precursors and one or more multi-functionalsurface ligands with a solvent to form a solution and heating thesolution to a suitable temperature, and in certain embodiments, methodsmay include the steps of admixing nanocrystal cores, one or morenanocrystal precursors, and one or more multi-functional surface ligandswith a solvent to form a solution and heating the solution to a suitabletemperature. In such embodiments, the one or more multi-functionalsurface ligands may at least include a nanocrystal binding center, alinker, and a functional group, which imparts functionality on thenanocrystal. As used herein the term “functional group” may refer to agroup which affects reactivity, solubility, or both reactivity andsolubility when present on a multi-functional surface ligand.Embodiments can include a wide variety of functional groups which canimpart various types of functionality on the nanocrystal includinghydrophilicity, water-solubility, or dispersibility and/or reactivity,and the functionality may generally not include only hydrophobicity oronly solubility in organic solvents without increasing reactivity. Forexample, a functional group which is generally hydrophobic but whichincreases reactivity such as an alkene or alkyne and certain esters andethers can be encompassed by embodiments, whereas alkyl groups, which donot generally impart reactivity but increase hydrophobicity may beexcluded.

In certain embodiments, the nanoparticles produced by the methods ofsuch embodiments may be coated with ligands which impart watersolubility and/or reactivity on the nanoparticle obviating the need forligand replacement. Without wishing to be bound by theory, eliminatingligand replacement may provide more consistent thermodynamic properties,which may lead to reduction in variability of coating and less loss ofquantum yield, among other improvements in the properties ofnanoparticles produced by the methods embodied herein. Eliminatingligand replacement may also allow for the production of nanoparticleshaving a wide variety of functional groups associated with the coating.In particular, while ligand replacement is generally limited toproduction of nanoparticles having amine and/or carboxylic acidfunctional groups, in various embodiments, the skilled artisan maychoose among numerous functional groups when preparing themulti-functional ligands and may, therefore, generate nanoparticleswhich provide improved water-solubility or water-dispersity and/orsupport improved crosslinking and/or improved reactivity with cargomolecules. See for example PCT Application Serial No. PCT/US09/59117filed Sep. 30, 2009 which are expressly incorporated herein by referenceas if set forth in full.

In some embodiments, the one or more signals indicative of nucleotideincorporation can be detected to permit visualization of polymeraseactivity. In some embodiments, such visualization happens in real timeor near real time. In some embodiments, the signal can be an opticallydetectable signal. In some embodiments, the optically detectable signalcan be a fluorescent signal or a nonfluorescent signal.

In some embodiments, one or more labels must be excited before they canbe visualized. In some embodiments, illumination of the reaction sitepermits observation of the signals, e.g., FRET signals, which indicatethe nucleotide incorporation event. In some embodiments, the opticalsystem comprises at least two elements, namely an excitation source anda detector. The excitation source generates and transmits incidentradiation used to excite the reactants contained in the array. Dependingon the intended application, the source of the incident light can be alaser, laser diode, a light-emitting diode (LED), a ultra-violet lightbulb, and/or a white light source. Where desired, more than one sourcecan be employed simultaneously. The use of multiple sources isparticularly desirable in applications that employ multiple differentreagent compounds having differing excitation spectra, consequentlyallowing detection of more than one fluorescent signal to track theinteractions of more than one or one type of molecules simultaneously.

The one or more signals indicative of nucleotide incorporation can bedetected and analyzed using any suitable methods and related devices.Typically, the optical system will achieve these functions by generatingand transmitting an incident wavelength to the polynucleotides isolatedwithin nanostructures, and collecting and analyzing the emissions fromthe reactants.

In some embodiments, detection is simplified by placing one or fewpolymerase and nucleic acid molecules in a sufficiently small volumesuch that background signal from non-incorporated nucleotides isminimized. This can be done, for example, by isolating, confining orimmobilizing a single polymerase, or a single template nucleic acidmolecule, in an optical confinement such as a zero-mode waveguide. Insome embodiments, the polymerase and/or template nucleic acid moleculecan be placed within a nanochannel. In some embodiments, the nanochannelcan be structured to permit isolation and/or elongation of individualnucleic acid molecules.

Exemplary labeling and detection strategies for use with one or more ofthe modified polymerases disclosed herein include, but are not limitedto, those disclosed in U.S. Pat. Nos. 6,423,551 and 6,864,626; U.S.Published Application Nos. 2005/0003464, 2006/0176479, 2006/0177495,2007/0109536, 2007/0111350, 2007/0116868, 2007/0250274 and 2008/08825. Awide variety of detectors are available in the art. Representativedetectors include but are not limited to optical readers,high-efficiency photon detection systems, photodiodes (e.g. avalanchephoto diodes (APD); APD arrays, etc.), cameras, charge couple devices(CCD), electron-multiplying charge-coupled device (EMCCD), intensifiedcharge coupled device (ICCD), photomultiplier tubes (PMT), a multi-anodePMT, and a confocal microscope equipped with any of the foregoingdetectors. Where desired, the subject arrays can contain variousalignment aides or keys to facilitate a proper spatial placement of eachspatially addressable array location and the excitation sources, thephoton detectors, or the optical transmission element as describedbelow.

In some embodiments, the detection system comprises: excitationillumination, optical transmission elements, detectors, and/orcomputers. In some embodiments, the detection system can compriseexcitation illumination which can excite the energy transfer or reportermoieties which produce a signal. The excitation illumination can beelectromagnetic energy, such as radio waves, infrared, visible light,ultraviolet light, X-rays or gamma rays. The source of theelectromagnetic radiation can be a laser, which possesses properties ofmono-chromaticity, directionality, coherence, polarization, and/orintensity. The laser can produce a continuous output beam (e.g.,continuous wave laser) or produce pulses of light (e.g., Q-switching ormode-locking). The laser can be used in a one-photon or multi-photonexcitation mode. The laser can produce a focused laser beam. Thewavelength of the electromagnetic radiation can be between about 325-840nm, or between about 325-752 nm, or between about 330-752 nm, or betweenabout 405-752 nm.

In some embodiments, the detection system comprises suitable opticaltransmission elements that are capable of transmitting light from onelocation to another with the desired refractive indices and geometries.The optical transmission elements transmit the excitation illuminationand/or the emitted energy in an unaltered or altered form. The opticaltransmission elements include: lens, optical fibers, polarizationfilters (e.g., dichroic filters), diffraction gratings (e.g., etcheddiffraction grating), arrayed waveguide gratings (AWG), opticalswitches, mirrors, dichroic mirrors, dichroic beam splitter, lenses(e.g., microlens and nanolens), collimators, filters, prisms, opticalattenuators, wavelength filters (low-pass, band-pass, or high-pass),wave-plates, and delay lines, or any combination thereof.

In some embodiments, spectral detection can also be combined and/orreplaced by other detection methods capable of discriminating betweenchemically similar or different labels in parallel, including, but notlimited to, polarization, lifetime, Raman, intensity, ratiometric,time-resolved anisotropy, fluorescence recovery after photobleaching(FRAP) and parallel multi-color imaging. See, for example, Lakowitz,supra. In the latter technique, use of an image splitter (such as, forexample, a dichroic mirror, filter, grating, prism, etc.) to separatethe spectral components characteristic of each label allows the samedetector, typically a CCD, to collect the images in parallel.

In some embodiments, multiple cameras or detectors can be used to viewthe sample through optical elements (such as, for example, dichroicmirrors, filters, gratings, prisms, etc.) of different wavelengthspecificity. Other suitable methods to distinguish emission eventsinclude, but are not limited to, correlation/anti-correlation analysis,fluorescent lifetime measurements, anisotropy, time-resolved methods andpolarization detection.

Suitable imaging methodologies that can be implemented for detection ofemissions include, but are not limited to, confocal laser scanningmicroscopy, Total Internal Reflection (TIR), Total Internal ReflectionFluorescence (TIRF), near-field scanning microscopy, far-field confocalmicroscopy, wide-field epi-illumination, light scattering, dark fieldmicroscopy, photoconversion, wide field fluorescence, single and/ormulti-photon excitation, spectral wavelength discrimination, evanescentwave illumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, multi-foci multi-photon, and/or other forms ofmicroscopy.

In some embodiments, the detection system can include one or moreoptical transmission elements that serve to collect and/or direct theincident wavelength to the reactant array; to transmit and/or direct thesignals emitted from the reactants to the photon detector; and/or toselect and modify the optical properties of the incident wavelengths orthe emitted wavelengths from the reactants. Illustrative examples ofsuitable optical transmission elements and optical detection systemsinclude but are not limited to diffraction gratings, arrayed wave guidegratings (AWG), optic fibers, optical switches, mirrors, lenses(including microlens and nanolens), collimators. Other examples includeoptical attenuators, polarization filters (e.g., dichroic filters),wavelength filters (low-pass, band-pass, or high-pass), wave-plates, anddelay lines. Examples of a suitable sequencing and detection systemsthat may be used according to the present disclosure include, forexample, U.S. Provisional Application Nos. 61/077,090, filed Jun. 30,2008; 61/089,497, filed Aug. 15, 2008; 61/090,346, filed Aug. 20, 2008;and 61/164,324, filed Mar. 27, 2009.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention mayhave been described in terms of specific examples or preferredembodiments, these examples and embodiments are in no way intended tolimit the scope of the claims, and it will be apparent to those of skillin the art that variations may be applied to the compositions and/ormethods and in the steps or in the sequence of steps of the methodsdescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLES Example 1: Production of a Polynucleotide Encoding an ExemplaryModified Polymerase

This example illustrates the production of a polynucleotide encoding anexemplary modified polymerase comprising the amino acid sequence of SEQID NO: 7. A nucleotide sequence encoding a B103 polymerase having theamino acid sequence of SEQ ID NO: 6 was synthesized and cloned into asuitable expression vector. The recombinant expression construct wastransformed into a suitable bacterial strain, and transformants werepicked and screened for expression of the recombinant protein. Themutations F383L and D384N were introduced via site-directed mutagenesisaccording to standard methods, thereby generating a nucleotide sequenceencoding a modified polymerase comprising the amino acid sequence of SEQID NO: 7 cloned into the expression vector pTTQ. FIG. 2 provides anexemplary depiction of this expression vector comprising an open readingframe comprising the amino acid sequence of SEQ ID NO: 7 (referred to inFIG. 2 as “B104”).

Example 2: Purification of an Exemplary Modified Polymerase

To obtain a purified preparation of the modified polymerase protein, ahighly expressing clone was selected and cultured to a cell density of0.5 OD at 37° C. The culture was shifted to 18° C. At OD 0.6, expressionof the recombinant protein was induced by the addition of IPTG to afinal concentration of 0.5 or 1 mM, and the culture was grown for anadditional 18 hours. Cells were harvested by differentialcentrifugation. The yield was approximately 5 g cells/liter. The cellpellets were frozen at −80° C. until processed. Unless otherwiseindicated, all subsequent steps were performed on ice or at 4° C. Topurify the modified polymerase, cell pellets were resuspended in 5-10 mllysis buffer (50 mM Tris pH 7.5, 50 mM glucose, 0.1 mM EDTA pH 8.0,0.05% Tween-20, 1 mM DTT) per gram of cell paste. The cells were lysedunder high pressure using a Microfluidizer, M110 (Microfluidics Corp,Boston, Mass.). Streptomycin sulfate (50% solution in 50 mM Tris pH 7.5)was added to the lysate to a final concentration of 2% while stirring onice. The lysate was stirred for 30 minutes. The cell debris was pelletedby centrifugation at 10,000 rpm in a SLC1500 rotor for 30 minutes. Thepellets were discarded. A 10% solution of polyethyleneimine (pH 7.5) wasadded to the lysate, drop wise, to a final concentration of 0.2% whilestirring on ice. The lysate continued stirring for 30 minutes and thencentrifuged as described previously. The supernatant was returned to theice bath. Solid ammonium sulfate was added to the stirring supernatantto a final concentration of 65% ammonium sulfate saturation (43grams/100 mls lysate). The sample was stirred for an additional onehour. The precipitated proteins were collected by centrifugation in anSS34 rotor for 30 minutes at 15,000 rpm. The supernatant was discarded.The precipitated protein pellets were stored at −20° C.

The pellets were also subjected to affinity purification using aHis-TRAP FF (GE Healthcare). One 5 ml His-TRAP FF column wasequilibrated in HIS Buffer A (25 mM Hepes pH 7.5, 500 mM NaCl, 1 mM DTT)and 5% His Buffer B (25 mM Hepes pH 7.5, 500 mM NaCl, 1 mM DTT, 500 mMimidazole pH 7.5) on an AKTA 10S (GE Healthcare). The ammonium sulfatepellets were resuspended in resuspension buffer (25 mM Hepes pH 7.5, 500mM NaCl, 1 mM DTT, 25 mM imidazole). Each sample was filtered with a 1μM filter (Acrodisc) and then applied to the His-TRAP column with a P960pump. The sample was eluted from the column with a 12-70 column volumegradient of His Buffer B ranging from 5% to 100%. 1.8 ml fractions werecollected. An aliquot of each fraction was subjected to SDS PAGE todetermine which fractions contained the purest expressed protein (SDSPAGE: 10% NuPage, MES).

The protein-containing fractions were pooled and diluted using HISBuffer A to a conductivity of 17 millisiemens/cm. The diluted solutionwas then subjected to affinity purification using a Heparin column. APoros HE50 column (10 mm×100 mm; 7.8 mls, Applied Biosystems) wasequilibrated in HE Buffer A (25 mM Hepes pH 7.5, 1 mM DTT, 0.05% Tween20) containing 10% HE Buffer B (25 mM Hepes pH 7.5, 1M NaCl, 1 mM DTT,0.05% Tween 20) on the AKTA10S. Fractions from the His-trap columncontaining the purest protein as judged by SDS PAGE, were pooled anddiluted with HE Buffer A to ˜12-20 mS/cm. The sample (˜250 ml) wasloaded onto the HE column at 10 mls a minute with the P960 pump. Theproteins were eluted from the column with a 20 column volume gradientfrom 10% to 100% HE Buffer B at flow rate of 8 ml/min (156 ml) and 4 mlfractions were collected. An aliquot of each fraction was subjected toSDS PAGE to determine the fractions that contained the purest fractionof the expressed protein (SDS PAGE: 10% NuPage, MES).

The purest protein samples (as determined by SDS PAGE) were pooled anddialyzed overnight against Storage Buffer (10 mM Tris pH 7.5, 100 mMNaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol). The concentration of thedialysates was then determined by UV spectroscopy. The dialyzed proteinwas diluted 1:10 or 1:20 in buffer containing 10 mM Tris pH 7.5, 0.1 mMEDTA, 100 mM NaCl; 25% glycerol. A UV wavelength scan from 220 nm to 320nm was performed using a DU780 spectrometer (Beckman). The proteinconcentration was determined from the average of three readings at 280nM. The molar extinction coefficient as determined by the Vector NTIpackage (Invitrogen, Life Technologies) is 98,130 M⁻¹ cm⁻¹ and thepredicted molecular weight was 67,717.

Example 3: Characterization of Extension Activity and NucleotideIncorporation Activity of Exemplary Reference and Modified Polymerases

The fractional extension activity of an exemplary modified polymerasehaving the amino acid of SEQ ID NO: 8 (referred to as “B104 (exo-)” inFIG. 3) was measured and compared to the fractional extension activitiesof two different exemplary reference polymerases, including an RB69reference polymerase comprising the amino acid sequence SEQ ID NO: 15and a His-tagged Phi-29 reference polymerase comprising the amino acidsequence of SEQ ID NO: 20 (referred to herein as “HP1 polymerase”). Allpolymerases were prepared according to the method of Example 1. Bothmodified and reference polymerases were evaluated and compared in anassay to measure the fractional extension activity of each polymerase,i.e., the fraction of nucleic acid templates that are extended by atleast one nucleotide in a polymerase reaction. In this assay, thefractional extension activity of a polymerase is defined as the fraction(measured as a percentage fraction) of nucleic acid templates that areextended by at least one nucleotide by the test or reference polymeraseunder the following reaction conditions: 50 mM Tris, pH 7.0, 50 mM NaCl,1 mM MnCl₂, 1 μM dNTP, 330 nM enzyme, 100 nM primer-template duplex and0.5% BSA, wherein the extension is performed at room temperature for 10seconds.

The primer-template duplex was formed by annealing the followingpolynucleotides:

(SEQ ID NO: 21) 5′-GGTACTAAGCGGCCGCATG-3′ (“TOP”)

This primer comprises a fluorescein moiety linked to the 5′ terminalnucleotide.

(SEQ ID NO: 22) 3′-CCATGATTCGCCGGCGTACTTTTTTT-5′ (“BOT T6T”)(SEQ ID NO: 23) 3′-CCATGATTCGCCGGCGTACAAAAAAA-5′ (“BOT A6A”)(SEQ ID NO: 24) 3′-CCATGATTCGCCGGCGTACCCCCCCC-5′ (“BOT C6C”)(SEQ ID NO: 25) 3′-CCATGATTCGCCGGCGTACGGGGGGG-5′ (“BOT G6G”)

Annealing of the primer of SEQ ID NO: 21 with any of the templates ofSEQ ID NOS: 22-25 results in formation of a non-extendible duplexcomprising a 7-nucleotide overhang.

The polymerase reaction products were resolved using PAGEelectrophoresis. The extended products were visualized by scanning thegel with a Bio-Rad Imager for fluorescence emissions at 488 nm. Theresults are depicted in FIG. 3. As shown in FIG. 3, the fractionalextension activity (as measured by both intensity and length ofproducts, as well as by reduced intensity of starting material) of amodified polymerase comprising the amino acid sequence of SEQ ID NO: 8was observed to be higher than the fractional extension activity of thereference RB69 polymerase comprising the amino acid sequence of SEQ IDNO: 15 and of the reference His-tagged Phi-29 polymerase comprising theamino acid sequence of SEQ ID NO: 20.

Example 4: Characterization of Exonuclease Activity of ExemplaryReference and Modified Polymerases

An assay comparing the endogenous exonuclease activity of a referencepolymerase, T7 DNA polymerase, with the exonuclease activity of apolymerase comprising the amino acid of SEQ ID NO: 7, was performed.Exonuclease activity was measured by incubating 2, 4 or 10 μg of eachenzyme sample with 7.5 μL Invitrogen Low Mass ladder, (10068-013) in a50 μL, reaction containing 10 mM Tris, pH 7.5, 50 mM NaCl and 10 mMMgCl₂ for 16 hours at 37° C. in an ABI 9700 PCR machine. The sampleswere resolved via electrophoresis on a 1.2% E-Gel (Life Technologies).The extent of exonuclease activity was estimated by visually assessingthe disappearance of the bands of the Low Mass ladder, as compared tonegative control reactions, to which water (“W”) or enzyme storagebuffer (“SB”) was added instead of purified polymerase). T7 DNApolymerase served as the reference polymerase (positive control,indicated as “+ control” in FIG. 4. The amount of protein added to eachreaction is indicated at the top of each well. (2, 4, 10 μg protein, orwater (“W”) or storage buffer (“SB”) for negative control lanes) Asshown in FIG. 4, both the reference and modified polymerases exhibitedcomparable levels of exonuclease activity.

To determine if the product contained single-strand DNase (ssDNase)activity that could result from contaminating polypeptides, the enzymewas tested with AMBION DNase Alert according to manufacturer'sinstructions (AM1970, Applied Biosystems). The fluorescently labeledDNase Alert substrate (suspended in TE) (10 μL), the supplied 10×buffer, and increasing concentrations of the test enzyme were combinedin a final volume of 100 μL. The samples were read in a MolecularDevices Plate reader at 485/590 excitation/emission for 60 min at 37° C.No detectable fluorescence was recorded. DNase I served as a positivecontrol. No ssDNase activity was observed in either control or samplereactions (results not shown). Exemplary results are depicted in FIG. 4.

Example 5: Characterization of Nanoparticle Tolerance of ExemplaryReference and Modified Polymerases

The nanoparticle tolerance of exemplary modified and referencepolymerases was measured and compared. The nucleotide incorporationactivity of an exemplary modified polymerase comprising the amino acidsequence of SEQ ID NO: 19 and further comprising the amino acidsubstitution D175A was measured both in the absence and presence ofequimolar amounts of a test nanoparticle. The results were compared tothose of an exemplary reference Phi-29 polymerase comprising the aminoacid sequence of SEQ ID NO: 20.

Primer extension activity was measured using an assay wherein freepyrophosphate released as a result of extension of a primer:templateduplex is detected as a fluorescent product. Reaction conditionsincluded 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM DTT, 100 nM polymerase,100 nM hairpin-template, and 2 mM MnCl₂ at 23° C., either with orwithout 100 nM of nanoparticle added to the mixture. Individualreactions were set up in microtiter plate wells and initiated by theaddition of 2 μl of 100 μM terminally-labeled nucleotide. The resultingfluorescence emissions were monitored as a function of time using aplate reader (Molecular Devices, SpectraMax M5). Reactions withoutnucleotides were also monitored as negative controls. Reactions weremonitored for 20 minutes or until the samples reached saturation.

Exemplary results are depicted in FIG. 5, which provides a graph ofrelative fluorescence observed in each sample (RFU, arbitrary units, Yaxis) against time (seconds, X axis). The various fluorescence tracesdepicted in FIG. 5 are as follows: solid circles and solid squares (twocurves along X axis): fluorescence traces of negative control reactionslacking polymerase and including or not including nanoparticles; opendiamonds: reaction including a reference Phi-29 polymerase comprisingthe amino acid sequence of SEQ ID NO: 1 and further comprising the aminoacid substitution N62D (Φ29 (exo-)) and including nanoparticles; opensquares and open circles: modified polymerase comprising the amino acidsequence of SEQ ID NO: 19, including and not including nanoparticles,respectively. Primer extension activity was measured as the averageslope of the curve obtained by plotting fluorescence (RFU) vs. time(seconds, X axis) between the zero time point and at 5 minutes. Theprimer extension activity in the presence and absence of dots wascomputed. Nanoparticle tolerance is determined as the percentage ofprimer extension activity, measured in the presence of testnanoparticle, relative to the amount of primer extension activityobserved in the absence of the test nanoparticle. These slope values foreach fluorescence trace are depicted below the graph in FIG. 5.

According to the results of FIG. 5, the modified polymerase of SEQ IDNO: 19 exhibits approximately 10% loss in primer extension activity inthe presence of equimolar amounts of the test nanoparticle (i.e., 90%nanoparticle tolerance under these defined reaction conditions), ascompared to a 50% loss in primer extension activity exhibited by thereference Phi-29 polymerase under identical test conditions (i.e., 50%nanoparticle tolerance exhibited by the Phi-29 polymerase under thesedefined reaction conditions).

Example 6: Characterization of Photostability of Exemplary Reference andModified Polymerases

The photostability of an exemplary reference polymerase comprising aHis-tagged version of Phi-29 polymerase (“HP1; see, e.g., U.S.Provisional Application No. 61/184,770, filed Jun. 5, 2009 fordisclosure of HP1 sequence and purification) comprising the amino acidsequence of SEQ ID NO: 20, and an exemplary modified polymerasecomprising the amino acid sequence of SEQ ID NO: 19 and furthercomprising the amino acid substitution D175A were characterized bymeasuring the amount of primer extension observed in each sample priorto and following exposure to excitation radiation at 405 nm. Reactions(100 μL) containing 50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 2 mMMnCl₂, 0.3% BSA, 10-200 nM polymerase and 100 nM nanoparticles (orpremade polymerase-nanoparticle conjugates; see, e.g., U.S. ProvisionalApplication No. 61/184,770 filed Jun. 5, 2009), and 100 nM5′-[³²P]-labeled templates or 5′-TAMRA-labeled templates were preparedwith and without the Oxygen Scavenging System (OSS). The OSS consists of0.1 mg/ml Glucose Oxidase (Sigma, Catalog #G3660-1CAP), 2 units/μlKatalase (Fluka, Catalog #02071), 2 mM Trolox, and 0.5% glucose (addedjust prior to illuminations with 405 laser). Aliquots (4 μL) were addedto a quartz cuvette having a path length of 1.5 mm and a height of 15 mm(Hellma, 105.252-QS) and illuminated with a 405 nm laser for a specifiedtime and at specified power levels. After illumination, samples wereremoved and placed on ice until extensions were performed at 23° C. Theextensions were performed by addition of 5 μM nucleotide hexaphosphatescomprising a hexaphosphate moiety linked to the 3′ carbon of the sugarmoiety, and further comprising a 6-carbon linker attached to theterminal phosphate but without any fluorescent label. Extensions wereperformed for 30 seconds followed by termination with loading buffer(90% formamide, 10 mM EDTA). Samples were resolved on denaturingpolyacrylamide gels (8M urea, 20% polyacrylamide) and exposed tophosphorimager screen. Representative results are provided in FIG. 6A.Polymerase activity was quantified as the % of extended primer (ascompared to the total starting amount of primer), as measured bydensitometric analysis. Phototoxicity is quantified by measuring thepercent decrease in polymerase activity that occurs when samples areilluminated compared to non-illuminated samples. The photostability isquantified by subtracting the observed photosensitivity from 100.

FIG. 6B shows the % activity retained (Y axis) after 30 seconds ofexposure to radiation of various intensities (X axis, W/cm²). As shownin FIG. 6B, the modified polymerase comprising the amino acid sequenceof SEQ ID NO: 19 and the amino acid substitution D175A (solid diamonds)retained 80-90% activity following exposure at 50 W/cm2, whereas thereference Phi-29 polymerase comprising the amino acid sequence of HP1(solid squares) retained less than 40% activity.

Example 7: Characterization of Values of Exemplary Reference andModified Polymerases

In this example, the t⁻¹ values of an exemplary modified polymerasecomprising the amino acid of SEQ ID NO: 8 and an exemplary referencepolymerase comprising the amino acid sequence of SEQ ID NO: 20 weremeasured and compared. These kinetic parameters were evaluated using anassay system wherein mixtures comprising a test polymerase and aprimer:template duplex were mixed in a stopped-flow assay withnucleotides. To measure t⁻¹, the primer used to form the primer:templateduplex was a non-extendible primer including a dideoxynucleotide at its3′ end, and the template was a synthetic DNA template comprising a donorAlexa Fluor 546 moiety at its 5′ end. This primer-template duplex waspreincubated with a test polymerase and a terminally-labeled nucleosidehexaphosphate or tetraphosphate (“ωdN4P” or “ωdN6P”, respectively) toform an E•DNA•ωdN4P or E•DNA•ωdN6P ternary complex. Theseterminally-labeled nucleotides comprise terminal-phosphate-labelednucleotides having an alkyl linker with a functional amine groupattached to the dye. The ternary complex was mixed with unlabelednucleotides in a stopped-flow experiment, and the resulting fluorescencewas monitored as described below. Unless noted otherwise, allconcentrations refer to the initial concentrations prior to mixing. Allexperiments were carried out in 50 mM Tris pH 7.5, 50 mM NaCl, 4 mM DTT,2 mM MnCl₂ and 0.2% BSA (“extension buffer”) using an AppliedPhotophysics SX20 stopped-flow spectrometer (Applied Photophysics,London, U.K.).

To prepare the primer:template duplex, 20 μM of primer and 20 μM oftemplate were mixed in solution. The solution was heated to 90° C. for 5minutes and then cooled stepwise in a thermocycler to 4° C. at a rate of1° C. every 18 seconds. Five different primer:template duplexes wereprepared, each comprising a different template sequence as describedbelow.

The nucleotide sequence of the primer was as follows:

(SEQ ID NO: 26) 5′-GCC TCG CAG CCG TCC AAC CAA CTC^(dd)C-3′where ^(dd)C indicates a dideoxycytosine triphosphate moiety. Thissequence is referred to as “dd-Top-25-mer”.

Four different templates were tested in separate assays. The nucleotidesequence of the first template was as follows:

(SEQ ID NO: 27) AF546-5′-CAG TAA CGG AGT TGG TTG GAC GGC TGC GAG GC-3′where AF546 indicates an Alexa Fluor 546 moiety. This sequence isreferred to as “dd-Template C-32-mer”.

The nucleotide sequence of the second template was as follows:

(SEQ ID NO: 28) AF546-5′-CAG TAA GGG AGT TGG TTG GAC GGC TGC GAG GC-3′where AF546 indicates an Alexa Fluor 546 moiety. This sequence isreferred to as “dd-Template G-32-mer”.

The nucleotide sequence of the third template was as follows:

(SEQ ID NO: 29) AF546-5′-CAG TAA AGG AGT TGG TTG GAC GGC TGC GAG GC-3′where AF546 indicates an Alexa Fluor 546 moiety. This sequence isreferred to as “dd-Template A-32-mer”.

The nucleotide sequence of the fourth template was as follows:

(SEQ ID NO: 30) AF546-5′-CAG TAA TGG AGT TGG TTG GAC GGC TGC GAG GC-3′where AF546 indicates an Alexa Fluor 546 moiety. This sequence isreferred to as “dd-Template T-32-mer”.

Annealing of the primer of SEQ ID NO: 25 with any of the templates ofSEQ ID NOS: 26-30 results in formation of a non-extendible duplexcomprising a 7-nucleotide overhang.

Preincubated mixtures comprising 200 nM of a primer:templex duplex, 660nM of test polymerase, and 14 μM of ωdN4P were prepared and then mixedwith a solution comprising the unlabeled cognate nucleotide (50 μM) in astopped-flow apparatus. The fluorescence at both 546 nm and 647 nm wasmonitored following mixing. The averaged stopped-flow fluorescencetraces were fitted with a single exponential function having the form ofEquation (1) to extrapolate the t⁻¹ value of the test polymerase. Usingthe above procedure, the estimated t⁻¹ values of an exemplary modifiedpolymerase comprising the amino acid sequence of SEQ ID NO: 8 and anexemplary reference polymerase comprising the amino acid sequence of SEQID NO: 1 were determined to be comparable (data not shown).

Example 8: Characterization of t_(po1) Values of Exemplary Reference andModified Polymerases

In this example, the t_(po1) values of an exemplary modified polymerasecomprising the amino acid of SEQ ID NO: 8 and was measured and comparedto the t_(po1) value of an exemplary reference polymerase comprising theamino acid sequence of SEQ ID NO: 20. These kinetic parameters wereevaluated using a stopped-flow assay wherein a mixture comprising anextendible dye-labeled primer:template duplex and test polymerase wasmixed with a solution comprising terminally-labeled nucleotidetetraphosphates or terminally-labeled nucleotide hexaphosphates. Theseterminally-labeled nucleotides comprise terminal-phosphate-labelednucleotides having an alkyl linker with a functional amine groupattached to the dye. The fluorescence of the resulting mixture wasmonitored over time. Unless noted otherwise, all concentrations refer tothe initial concentrations prior to mixing. All experiments were carriedout in 50 mM Tris pH 7.5, 50 mM NaCl, 4 mM DTT, 2 mM MnCl₂ and 0.2% BSA(“extension buffer”) using an Applied Photophysics SX20 stopped-flowspectrometer (Applied Photophysics, London, U.K.).

The primer-template duplex comprised an extendible primer annealed to asynthetic DNA template comprising a donor Alexa Fluor 546 moiety at its5′ end. To prepare the primer:template duplex, 20 μM of primer and 20 μMof template were mixed in solution. The solution was heated to 90° C.for 5 minutes and then cooled stepwise in a thermocycler to 4° C. at arate of 1° C. every 18 seconds.

The nucleotide sequence of the primer was as follows:

(SEQ ID NO: 31) 5′-GTT GCA AAG GAG CGG GCG-3′

Four different templates were prepared and tested in separate assays.The nucleotide sequence of the first template was as follows:

(SEQ ID NO: 32) AF546-5′-CGT TCC CCG CCC GCT CCT TTG CAA C-3′where AF546 indicates an Alexa Fluor 546 moiety.

The nucleotide sequence of the second template was as follows:

(SEQ ID NO: 33) AF546-5′-CGT TCC GCG CCC GCT CCT TTG CAA C-3′where AF546 indicates an Alexa Fluor 546 moiety.

The nucleotide sequence of the third template was as follows:

(SEQ ID NO: 34) AF546-5′-CGT TCC ACG CCC GCT CCT TTG CAA C-3′where AF546 indicates an Alexa Fluor 546 moiety.

The nucleotide sequence of the fourth template was as follows:

(SEQ ID NO: 35) AF546-5′-CGT TCC TCG CCC GCT CCT TTG CAA C-3′where AF546 indicates an Alexa Fluor 546 moiety.

Annealing of the primer of SEQ ID NO: 25 with any of the templates ofSEQ ID NOS: 26-29 results in formation of an extendible duplexcomprising a 7-nucleotide overhang.

Each primer-template duplex (200 nM) was preincubated with testpolymerase (660 nM) and to form an E•DNA binary complex. This binarycomplex was mixed with the cognate omega-labeled nucleosidetetraphosphate (ωdN4P; 14 μM) in a stopped-flow experiment, and thefluorescence at both 546 nm and 647 nm was monitored following mixing.The averaged stopped-flow fluorescence traces were fitted with thedouble exponential function of Equation (2) to extrapolate the t_(po1)value of the test polymerase:

Using the above procedure, the estimated t_(po1) values of an exemplarymodified polymerase comprising the amino acid sequence of SEQ ID NO: 8and an exemplary reference polymerase comprising the amino acid sequenceof SEQ ID NO: 1 were determined to be comparable (data not shown).

Example 9: Kinetics of Nucleotide Incorporation by Reference andModified Polymerases with Different Nucleotide Compounds

In this example, the kinetics of nucleotide incorporation by exemplaryreference and modified polymerases were analyzed and compared in astopped-flow nucleotide incorporation reaction comprising the testpolymerase of interest, labeled nucleotide polyphosphates and anextendible primer:template duplex labeled with ALEXA FLUOR 546(“AF546”). The AF546-labeled primer-template duplex was preincubatedwith the test polymerase, and then mixed in a stopped-flow assay withterminally-labeled nucleotides comprising a dye label attached to theterminal phosphate group using identical reaction conditions as used fort_(po1) measurements in the preceding Example (Example 8, above).

In one study, the primer:template duplex was formed using a primerhaving SEQ ID NO: 31 and a template having SEQ ID NO: 32. Three separateassays were conducted, each using a mutant Phi-29 polymerase and anysingle one of the following three different terminally labelednucleotides: AF647dG3P, AF647dG4P, AF647dG6P. These nucleotides comprisean ALEXA FLUOR dye (“AF647”) on the terminal phosphate, but differ fromeach other in the number of phosphates included in the polyphosphatechain. The resulting fluorescence time traces are depicted in FIG. 7A,reaction comprising AF647dG3P; FIG. 7B, reaction comprising AF647dG4P;FIG. 7C, reaction comprising AF647dG6P.

In another study, the fluorescence time traces observed using twodifferent Phi-29 mutant polymerases were obtained and compared. Theresults are depicted in FIGS. 8A and B.

As illustrated by FIGS. 7A, B and C, and FIGS. 8A and B, differentpolymerase-nucleotide combinations result in different time traces offluorescence emission. By testing various polymerase-nucleotidecombinations, it is possible to select combinations that exhibit optimalnucleotide binding affinities and binding speeds, as well as optimalamplitudes of fluorescence change during the nucleotide incorporationreaction so as to optimize the detectability of the reaction progress.

Example 10: Real-Time Single Molecule Sequencing Reaction Using anExemplary Modified Polymerase and Labeled Nucleotides

A exemplary modified polymerase comprising the amino acid sequence ofSEQ ID NO: 8 was used in a nucleotide incorporation reaction comprisingdonor-labeled template and acceptor-labeled nucleotides. A graphicaldepiction of this nucleotide incorporation reaction system, showing theimmobilized donor-labeled template, test polymerase and acceptor-labelednucleotides, is provided in FIG. 9.

Preparing PEG-Biotin Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and biotin-PEG to produce a low densitybiotin surface with a PEG coating to prevent non-specific background ofproteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of containing approximately 2 μl of fluid.

Attaching Biotinylated DNA to Low Density PEG-Biotin Surfaces:

Streptavidin protein was diluted to 200 μM in Incubation Buffer (50 mMNaCl; 50 mM Tris-Cl pH=7.5; 0.5% BSA was optionally added in someexperiments). Diluted streptavidin was flowed into fluidic chamber andstreptavidin was incubated for 10-15 minutes. Chambers were washed 1×with 1 ml Incubation Buffer. Biotinylated-DNA templates were diluted to10 μM in Incubation Buffer and allowed to bind for 10-15 minutes.Surfaces were washed 1× with 1 ml Incubation Buffer.

The primer sequence was as follows:

(SEQ ID NO: 36) 5′-GCCTCGCAGCCGTCCAACCAA CTCC-3′

The template sequence was as follows:

(SEQ ID NO: 37) Cy3-5′-TGCCACCGGAGTTGGT TGGACGGCTGCGAGG C-3′- Biotin

The extension sequence for a duplex formed between this primer andtemplate in the presence of guanine- and thymine-comprising nucleotidesis GGTGG.

A reaction mix of 100 uL was prepared comprising 200 nM dye-labeledguanine nucleotide hexaphosphates, which included the dye AF647 attachedto the omega-phosphate group (AF647-w-dG6P); 200 nM unlabeled thyminenucleotide tetraphosphates (dT4P); and 200 nM of test polymerase inextension buffer (50 mM ACES, pH 6.5; 50 mM NaCl) supplemented with 0.4%Glucose, 0.1 mg/mL Glucose Oxidase, 2000 unit/mL Katalase, 2 mM Troloxand 2 mM MnCl₂.

This reaction mixture was injected into the fluidic chamber comprisingimmobilized biotinylated duplex and the surface was imaged by excitingthe fluorescence donor at a power density of about 100 W/cm² at 532 nm.Images were recorded using an Olympus microscope outfitted with a TIRFobjective lens (100×; 1.45 NA). Emissions were imaged on a CCD camera.Images were collected at a frame rate of approximately 30 ms/frame.

To convert the observed fluorescence emissions detected during thenucleotide incorporation reaction into nucleotide sequence information,the raw data comprising a movie of observed emissions was firstprocessed by using a Hidden Markov Model (HMM)-based algorithm to detectand identify FRET events. The subsequent detected FRET events werefiltered and filtered sequences were aligned. Each of these two steps,FRET event detection and sequence analysis, are described in more detailbelow. The HMM-based algorithm was used to analyze the data.

1) Detection of FRET Events

The analysis underlying FRET event detection is designed to processspatially correlated movie(s) comprising real time sequence fluorescenceemission data, and extract time-series of interest from those data. Amovie typically contains one or more channels where each channelrepresents the same spatial location at different wavelengths. Theanalysis chain begins with the submission of one or more movies to theanalysis machine via a comprehensive user interface. The user interfacerequires the user to input various parameters which describe themovie(s) (e.g. channel regions, dye emission properties). Once this datais submitted the movie(s) are then processed by the image analysissoftware where a sliding window of N frames propagates through the moviecalculating a temporal local average of the frames within the window. Ateach position of the window in the movie, the local average image isthen further processed and enhanced using well known image processingalgorithms and a record of the maximum projection of all the localaverage images is recorded to produce a global image of the movie. Thisglobal image is the input into a spot identification algorithm whichproduces a set of spots identified by a unique spot identification, itsx and y location and its corresponding channel Each set of spots for agiven channel is then registered to the set of spots in every otherchannel. In this way a set of spot tuples is constructed. If a detectedspot in one channel does not have a corresponding detected spot inanother channel, then the position of the undetected spot using thetransformation between the two channels and the location of the detectedspot is inferred. Once a complete set of spot tuples is constructed themovie is iterated over and at each frame the amplitude of each spot iscalculated and appended to the appropriate time-series.

The collection of time-series from a spot tuple consists of time-seriesfrom donor and corresponding acceptor channels. This collection iscalled a Vector Time-Series (VTS). The FRET detection process startswith a data segmentation step using a Markov Chain Monte-Carlo (MCMC)algorithm. Each segment of VTS is modeled by a multivariate Gaussianmodel, with each of the channel modeled by a mean and a standarddeviation. This model establishes a baseline for each channel, fromwhich quantities such as “Donor Down” and “Acceptor Up” can becalculated. A Hidden Markov Model (HMM) was used to model the observeddata. The underlying states consist of a null state, a blink state and anumber of FRET states (one for each acceptor channel). Each state hasits emission probability, which reflects the state's correspondingphysical concept. FRET states are characterized by significant “donordown” and “acceptor up” signals. Blink state is characterized bysignificant “donor down” with no “acceptor up”. Null state ischaracterized by no “donor down” and no “acceptor up”. Given theobserved VTS signal, the emission matrix, and a state transitionprobability matrix, the most probable state path can be computed usingthe Viterbi algorithm. This state path assigns each of the frames to astate. Temporally neighboring FRET frames are grouped into FRET events.For each of the detected FRET events, a list of event features arecalculated, including event duration, signal average, signal to noiseratio, FRET efficiency, probability of event, color calling and otherfeatures. This list of events and corresponding features are stored in afile.

The final stage of the automated analysis generates a report summarizingthe results in the form of a web page containing summary image,statistics of the spots and FRET detection, together with line intensityplots and base call plots. See for example, Watkins et al., “Detectionof Intensity Change Points in Time-Resolved Single-MoleculeMeasurements” J. Phys. Chem. B., 109(1):617-628 (2005).

Using the above process, the movie data obtained from the sequencingreactions was analyzed to detect and identify FRET events according tothe process described above. The FRET events were then processed toidentify sequences as described below.

Sequence Analysis

Beginning with the set of detected Forster resonance energy transfer(FRET) events, a data overview was constructed in the form of a colorimage interpreted as a sequencing plot. To generate the plot, theoriginal FRET event data was pre-processed using a set of filtersconstructed by a priori knowledge of the sequence. For each reactionsite (each molecule) an ordered sequence of FRET events was constructed.The base call letters for each FRET event (e.g. “A”, “C”, “G” or “T”)were concatenated to form a sequence ASCII string. The order of lettersin the string reflects the temporal relationship of the events. Giventhat the expected sequence was known a priori, a regular expression wasthen constructed which represented the full or partial expected sequenceor sequence pattern. Matching against the regular expression (expectedsequence) was then computed for each sequence in the set and the startand stop indices of the match were recorded. A color plot image was thenconstructed where each row corresponds to a sequence in the set. Theplot image was padded to accommodate sequences of different lengths. Acolor map of 2*N+1 colors was constructed, where N denotes the number ofpossible base calls in each sequence (N=2 for the plot of this Example).N colors were assigned to the base characters which fell within thepattern, N colors were assigned to the base characters which did notfall within the pattern (muted color), and finally a color was assignedto the padding (background) of the image. The rows of the image werethen sorted according to the number of base calls in the first part ofthe sequence pattern. The rows of the image were also aligned such thatthe start of the expected sequence is in the same column for all rows ofthe plot.

One representative output using the reaction conditions described aboveis depicted in FIG. 10.

Example 11: Detecting Nucleotide Incorporation with Labeled BiomoleculeConjugates Comprising a Modified B103 Polymerase Linked to a FluorescentDye Label

In this Example, a modified B103 polymerase comprising the amino acidsequence of SEQ ID NO: 42, below, was prepared, biotinylated and labeledas outlined below. This modified B103 polymerase comprises the aminoacid sequence of SEQ ID NO: 8 and further includes the mutation H370R aswell as a biotinylation site and His tag fused to the N-terminus of theprotein. The dye-labeled polymerase conjugate was then used to studynucleotide incorporations in single molecule format.

Preparation of Biotinylated Polymerase

The construct HB B104 (H370R)_pAN6 was transformed and expressed inCVB101 (for in vivo biotinylation) cells. The cells were grown at 30° C.to OD 0.6 and induced with 0.5 mM IPTG. Upon induction 200 uM D-Biotinwas added and cultures were moved to 18° C. shaker and grown O/N andharvested the following morning. Cell pellets were resuspended in BufferB and sonicated to lyse. PEI (0.3%) was added to cell resuspension andincubated on ice for 30 min. Cell resuspension was centrifuged to removecell debris and DNA. Ammonium sulfate was added to cell lysate at finalconcentration of 55%. Lysate was centrifuged and pellets containing HBB104 (H370R) were resuspended in Buffer, loaded onto EMD-sulfate columnand eluted with linear gradient 10-100% BufferB. Fractions containing HBB104 (H370R) were pooled and loaded onto a His Trap column, eluted withlinear gradient from 5-100% Buffer C. Peak fractions were pooled andloaded onto a Heparin column, eluted with a linear gradient from 10-100%B. Fractions were then quantitated and analyzed for polymerase activity.

Buffer Compositions were as Follows:

Buffer A: 30 mM Tris pH 7.5, 100 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 8%glycerol

Buffer B: 30 mM Tris pH 7.5, 1 M NaCl, 2 mM DTT, 0.1 mM EDTA, 8%glycerol

Buffer C: 30 mM Tris pH 7.5, 100 mM NaCl, 2 mM DTT, 0.5M Imidazole, 8%glycerol.

Preparing NHS-Ester Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and NHS-ester to produce a low densityNHS-ester surface with a PEG coating to prevent non-specific backgroundof proteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of carrying approximately 2 μl of fluid.

Attaching Amine Terminated Hairpin DNA to Low Density NHS-EsterSurfaces: Target DNA hairpin sequence:

5′-TTTTTTTTACCCCCGGGTGACAGGTTXTTCCTGTCACCC-3′(SEQ ID NOS 38 and 49, respectively, in order of appearance)where “X” is an amine group.

The target DNA was diluted to 500 nM in 1 M NaHCO₃. The diluted targetmolecules were flowed into the fluidic chamber and incubated for 1 hour.Chambers were washed 1× with 1 ml deactivating buffer (ethanolamine).Surfaces were washed 1× with 1 ml incubation buffer (50 mM Tris-Cl,pH=7.5; 50 mM NaCl; 0.3% BSA).

SA-Polymerase Conjugate Preparation:

Streptavidin was labeled with Cy3. Streptavidin-Cy3 was mixed withbiotinylated mutant B103 polymerase (b-B103-exo minus) comprising theamino acid sequence of SEQ ID NO: 42 at a 1:1 ratio ofSA-protein:b-B103-exo minus in 1×PBS to produce conjugates comprisingbiotinylated mutant B103 polymerase linked to dye.

Briefly, 500 μl of a 3.4 μM solution of Cy3 streptavidin (Invitrogen,SA1010) was mixed with 25 μl of 200 μM biotin-B104 H370R. Twenty fivemicroliters of 5M NaCl were added to the mixture and it was left at 4deg C. for 1 hour. To remove any free, unconjugated labeledstreptavidin, the mixture was diluted with an equal volume of phosphatebuffer saline buffer (PBS) and loaded onto a 1 ml HisTrap cartridge (GEHealthcare). Following the loading, the cartridge was washed with PBSuntil the initially colored eluate from the cartridge became completelycolorless. Finally, the bound Cy3 streptavidin—biotin B104 H370Rconjugate was eluted off the cartridge with a solution of 500 mMimidazole in PBS buffer containing 200 mM additional NaCl. To the elutedmaterial was added 50 mM biocytin to a final concentration of 5 mM, andthe mixture was dialyzed overnight against a solution containing 50%glycerol, 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM DTT.

SA-Cy3-b-B103 Binding to Templates:

The conjugates were diluted to 1 nM in binding buffer (50 mM Tris-Cl;pH=7.5; 0.3% BSA; 100 mM NaCl). The conjugates were flowed into thefluidic chamber which were previously loaded with DNA templates on thesurface. Surfaces were incubated for 5 minutes with conjugates. Surfaceswere washed with 1×1 ml incubation buffer.

Fluorescence Imaging:

The microscope body was purchased from Olympus and was outfitted with aTIRF objective lens (100×; 1.45 NA). The excitation light passes throughan excitation filter (EX FT—543/22), and dichroic mirror (DM—532) andthe sample was epi-illuminated (Coherent) using TIR at typically 100W/cm². Upon excitation, the resulting epifluorescence emission passedthrough an emission filter (EM FT—540LP) and the resulting emission wassplit into three paths (tri-view) using 2 dichroic mirrors and theappropriate bandpass filters for the dye sets of choice. Using thisfilter combination, we were able to spectrally resolve 1 donor dye and 3acceptor dyes in 3 detection channels.

In separate experiments, 1 donor dye and 4 different acceptor dyes couldbe resolved in 4 detection channels. The optical detection scheme was asfollows: DC1=635, F1 640LP; DC2=675, F2=688/31; DC3=705, F3=700 LP. Thedonor dye used in this case was CY3 and the 4 acceptor dyes are asfollows DY634, AF647, AF676, AF700

The emissions resulting in each experiment were imaged on a CCD camera.Images were collected at a frame rate of approximately 20 ms.

Three-Color Nucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with conjugate bound toDNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates. In this example, the synthesized strandis expected to have the following sequence: (G)₅T(A)₈ (SEQ ID NO: 50).Terminal phosphate-labeled nucleotides and 125 nM cold dC6P were usedfor the nucleotide incorporation reaction. The labeled nucleotidesincluded 125 nM 647-dT6P, 125 nM 676-dG6P, 125 nM 700-dA6P. The spectralsignatures for the ALEXA FLUOR-676 G signal, AF-647 T signal, and AF-700A signal were identified that resulted from fluorescence resonanceenergy transfer (FRET) from the Cy3 donor molecule, and corresponded tothe correct insertion sequence pattern.

Analysis of Three-Color Sequencing Results

Resulting pattern sequencing data was processed using an alignmentalgorithm. The alignment algorithm found 100 molecules in the field ofview, which demonstrated completion of the full 14-nucleotide sequence((G)₅T(A)₈, which represented approximately 20% of the total singlemolecule donor population. The consensus sequence was determined usingan HMM alignment algorithm (e.g., see Example 14). By plotting theaccuracy definition (measured as a percentage value) against the HMMscore (X axis), a linear relationship was detected (data not shown).Various measurements of accuracy can be devised that can be suitable forsuch analysis. In one exemplary experiment, the accuracy was estimatedaccording to the following equation:

${\alpha( {T,A} )} = \frac{\beta - \delta - \eta + \lambda}{2\lambda}$

The measurement of accuracy in the above equation is intended to providesome measure of similarity between some given template, T, and somealignment, A, of an observed sequence O. It should be noted thatalphabet of T, A, and O are identical. The length of T is denoted by λ,the number of deletions in the alignment A by δ, the number ofinsertions in the alignment by η, and the number of matches in thealignment by β. Equation (1) is normalized by λ such that a an accuracyof 1 indicates a total agreement, and an accuracy of 0 indicates noagreement between T and A. The above definition of accuracy is providedas an example only and is in no way intended to limit the disclosure toany particular theory or definition of accuracy; alternative definitionsof accuracy are also possible and it may be suitable to use suchalternative definitions in some contexts.

The accuracy in this system using an HMM alignment threshold of 0 wasestimated to be approximately 80% (data not shown).

Four-Color Nucleotide Incorporation Reaction:

Oligonucleotides 401 template molecule: (SEQ ID NO: 39)TTTTTCCCCGACGATGCCTCCCC g ACA Cgg Agg TTC TAT CATCgT CAT CgT CAT CgT CAT Cg- Biotin TEG-T-3 Primer for 401 template:(SEQ ID NO: 40) 5′ TGA TAG AAC CTC CGT GTC 3′

In this example, the synthesized strand is expected to have thefollowing sequence: GGGGAGGCATCGTCGGGAAAA (SEQ ID NO: 41)

Nucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with SA-Cy3-b-B103 boundto DNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates.

The terminal phosphate-labeled nucleotides used for the nucleotideincorporation reaction included 125 nM DY634-dA6P, 125 nM 647-dT6P, 125nM 676-dG6P, 125 nM 700-dC6P. The spectral signatures for the DY-634 Asignal, and the ALEXA FLUOR G, T and C signals (AF-676 G signal, AF-647T signal, and AF-700 C signal) were identified that resulted fromfluorescence resonance energy transfer (FRET) from the Cy3 donormolecule, and corresponded to the correct insertion sequence pattern.4-color sequence alignment was obtained by visual inspection.

The observed FRET event durations for various SA-Cy3-b-B103 conjugates,the event count distributions, and the observed extension speeds ofvarious SA-Cy3-b-B103 conjugates were calculated.

The amino acid sequence of the modified B103 polymerase comprising themutation H370R and a biotinylation site and His tag fused to itsN-terminus was as follows:

(SEQ ID NO: 42)         10         20         30         40MSHHHHHHSM SGLNDIFEAQ KIEWHEGAPG ARGSKHMPRK        50         60         70         80MFSCDFETTT KLDDCRVWAY GYMEIGNLDN YKIGNSLDEF        90        100        110        120MQWVMEIQAD LYFHNLKFDG AFIVNWLEHH GFKWSNEGLP       130        140        150        160NTYNTIISKM GQWYMIDICF GYKGKRKLHT VIYDSLKKLP       170        180        190        200FPVKKIAKDF QLPLLKGDID YHAERPVGHE ITPEEYEYIK       210        220        230        240NAIEIIARAL DIQFKQGLDR MTAGSDSLKG FKDILSTKKF       250        260        270        280NKVFPKLSLP MDKEIRRAYR GGFTWLNDKY KEKEIGEGMV       290        300        310        320FDVNSLYPSQ MYSRPLPYGA PIVFQGKYEK DEQYPLYIQR       330        340        350        360IRFEFELKEG YIPTIQIKKN PFFKGNEYLK NSGAEPVELY       370        380        390        400LTNVDLELIQ EHYEMYNVEY IDGFKFREKT GLFKEFIDKW       410        420        430        440TYVKTREKGA KKQLAKLMLN SLYGKFASNP DVTGKVPYLK       450        460        470        480EDGSLGFRVG DEEYKDPVYT PMGVFITAWA RFTTITAAQA       490        500        510        520CYDRIIYCDT DSIHLTGTEV PEIIKDIVDP KKLGYWAHES       530        540        550        560TFKRAKYLRQ KTYIQDIYAK EVDGKLIECS PDEATTTKFS       570        580        590        600VKCAGMTDTI KKKVTFDNFR VGFSSTGKPK PVQVNGGVVL VDSVFTIK

Example 12: Measurement of t⁻¹ and t_(po1) Values of Modified Phi-29 andB103 Polymerases

In this example, the t⁻¹ and t_(po1) values of a Phi-29 polymerasecomprising the amino acid sequence of SEQ ID NO: 1 and a modified B103polymerase comprising the amino acid sequence of SEQ ID NO: 8 (referredto as “mB103 in the table below) and further including amino acidsubstitutions at various positions were measured using a stopped-flowprocedure. The stopped-flow techniques for measuring t_(po1) (1/k_(po1))followed the techniques described by MP Roettger (2008 Biochemistry47:9718-9727; M. Bakhtina 2009 Biochemistry 48:3197-320).

Stopped-Flow Measurements of t_(po1)

Template C sequence: (SEQ ID NO: 44) 5′-CGTTAACCGCCCGCTCCTTTGCAAC-3′Primer sequence: (SEQ ID NO: 45) 5′-GTTGCAAAGGAGCGGGCG-3′

The template sequence (SEQ ID NO: 44) further included an Alexa Fluor546 dye moiety bonded to the 5′ position of the template.

The kinetics of nucleotide incorporation by each polymerase was measuredin an Applied Photophysics SX20 stopped-flow spectrometer by monitoringchanges in fluorescence from the dye-labeled primer-template duplexcomplexed to enzyme, following the mixing of the enzyme-DNA complex withdye-labeled nucleotides. These dye-labeled nucleotides compriseterminal-phosphate-labeled nucleotides having an alkyl linker with afunctional amine group attached to the dye, and have the generalstructure shown in FIG. 11. This structure includes a sugar bonded to ahexaphosphate chain at the 5′ carbon position, and to a nucleotide base(denoted as “N”). The terminal phosphate group of the hexaphosphate islinked to a 6-carbon linker, and the other end of the 6-carbon linker isattached to a dye moiety (denoted as “dye”), typically through an amidebond. In this example, the particular dye-labeled nucleotide added was alabeled nucleotide hexaphosphate comprising a guanine base at the N(base) position and an Alexa Fluor 647 (AF647) at the dye position, andis referred to herein as “AF647-C6-dG6P”.

The primer and template were annealed to form a dye-labeledprimer-template duplex using standard methods. This duplex waspreincubated with polymerase. The mixture included 330 nM recombinantDNA polymerase, 100 nM template/primer duplex in buffer (“reactionbuffer”) comprising 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM DTT, 0.2%BSA, and 2 mM MnCl₂. The dye-labeled nucleotide AF647-C6-dG6P was thenadded to a final concentration of 7 μM, and the resulting fluorescencewas monitored over time.

The averaged (5 traces) stopped-flow fluorescence traces (>1.5 ms) werefitted with a double exponential equation (1) to extrapolate the ratesof the nucleotide binding and product release,Fluorescence=A ₁ *e ^(−k) ¹ ^(*t) +A ₂ *e ^(−k) ^(po1)^(*t)+C  (equation 1)where A₁ and A₂ represent corresponding fluorescence amplitudes, C is anoffset constant, and k1 and kpo1 are the observed rate constants for thefast and slow phases of the fluorescence transition, respectively.Stopped-Flow Measurements of t⁻¹

The stopped-flow techniques for measuring t⁻¹ (1/k⁻¹) followed thetechniques described by M. Bakhtina (2009 Biochemistry 48:3197-3208).

Template C sequence: (SEQ ID NO: 46)5′-CAGTAACGG AGT TGG TTG GAC GGC TGC GAG GC-3′ Dideoxy-primer sequence:(SEQ ID NO: 47) 5′-GCC TCG CAG CCG TCC AAC CAA CTC ddC-3′

The rate of the nucleotide dissociation (k⁻¹) from the ternary complexof [enzyme•DNA•nucleotide] was measured in an Applied Photophysics SX20stopped-flow spectrometer by monitoring changes in fluorescence from influorescence from a duplex Alexa fluor 546 dye-labeled-DNA templatefollowing the mixing of the [enzyme•DNA•labeled nucleotide] ternarycomplex with 50 μM cognate non-labeled deoxynucleoside triphosphate in abuffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM DTT, 0.2%BSA, and 2 mM MnCl₂.

The ternary complexes were prepared using: 330 nM polymerase, 100 nMtemplate/primer duplex, and 7 μM terminal phosphate-labeled nucleotides(AF647-C6-dG6P).

The averaged stopped-flow fluorescence traces (>1.5 msec) were fittedwith a single exponential equation (2) to extrapolate the rate of thenucleotide dissociation (k⁻¹) from the [enzyme•DNA•nucleotide] ternarycomplex.Fluorescence=A ₁ *e ^(−k) ⁻¹ ^(*t)+C  (equation 2)where A₁ represents the corresponding fluorescence amplitude, C is anoffset constant, and k⁻¹ and the observed rate constants for thefluorescence transition.

Some representative results of the stopped flow data are shown in thetable below.

TABLE Summary of t_(pol) and t₋₁ measurements for various exemplarymodified Phi-29 and B103 polymerases Protein t_(pol) t₋₁ mB103 (SEQ ID:8) 14 16 mB103 + H370R 17 43 mB103 + H370Y 15 12 mB103 + E371R 11 17mB103 + E371Y 11 7 K372R 14 12 K380R 783 17 mB103 + D507G 11 13 mB103 +D507H 7 16 mB103 + K509Y 10 20 Phi-29 (exo-) 11 27 Phi-29 (exo-) + T373R15 81 Phi-29 (exo-) + T373Y 14 45

Example 13: Measurement of Primer Extension Activity of a SamplePolymerase Using a Fluorescein-Labeled Oligonucleotide

This example provides an exemplary assay for primer extension activityin a sample. Primer extension activity is quantified by monitoring thefluorescence intensity change over time during extension of afluorescein-labeled hairpin oligonucleotide, comprising the followingnucleotide sequence, known as “oligo 221” (SEQ ID NO: 43). Thefluorescence intensity correlates with the level of primer extensionactivity in the sample.

(SEQ ID NO: 43) (5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCA-CC(fluorescein-T)GC-3′).

The extension reactions are performed in 1× extension buffer (50 mM Trisbuffer pH 7.5, 50 mM NaCl, 10 mM MgCl₂ and 0.5 mM MnCl₂). To reactionwells that contain 100 μL of 150 nM of a fluorescein-labeled hairpinoligonucleotide, oligo221 (SEQ ID NO: 43, above) and 10 nM of polymerase(or conjugated polymerase) in extension buffer, 2 μL of 1 mM dATP (finalconcentration: 20 μM) is added to initiate the enzymatic reaction andthe fluorescence intensity in each well is recorded at 525 nmfluorescence with 490 nm excitation for every 20 seconds for the next 10minutes. Control reaction wells include the same components without anyaddition of dATP. The fluorescence intensity at 525 nm (as measured inarbitrary fluorescence units, RFU, y axis) is plotted against time(seconds, X axis) for each sample, as well as the control wells (nonucleotide). The fluorescence time course data from each well is used tocalculate the primer extension activity of each sample using thefollowing equations:

${{Activity}( {{base}\text{/}\sec\text{/}{enz}} )} = {{\frac{\Delta\;{RFU}_{{{sample}\mspace{14mu}}_{—}}{per}_{—}\sec}{\Delta\;{RFU}_{{\max\mspace{14mu}}_{—}}{per}_{—}{nMsubs}} \times \frac{1}{50\mspace{14mu}{nM}} \times 7({base})\mspace{14mu}{and}\mspace{14mu}\Delta\;{RFU}_{{\max\mspace{14mu}}_{—}}{per}_{—}{nMsubs}} = \frac{{RFU}_{\max} - {RFU}_{\min}}{{substr}_{—}{{conc}.({nM})}}}$

Where: RFU_(max) is the average maximal RFU in the reference polymerasereaction wells; RFU_(min) is the average minimal RFU in the referencepolymerase control wells; Substr_conc. (nM) is the oligo 221concentration in assay, which is 150 nM; and:

${\Delta\;{RFU}_{{sample}_{—}}{per}_{—}\sec} = \frac{{RFU}_{t} - {RFU}_{0}}{t( \sec )}$

Where: t (sec) is the time period where the fluorescence intensityincreases in the reference enzyme reaction well linearly from the start;RFU_(t) is the average RFU of the reference enzyme extension wells at tsecond point; and RFU₀ is the average RFU of the reference enzymeextension wells at the start point.

Example 14: Preparation of Core-Shell Nanoparticle CdSe/4CdS-3.5ZnS

Core Synthesis

Cores are prepared using standard methods, such as those described inU.S. Pat. No. 6,815,064, the only change being that the growth is haltedat 535 nm emission. These cores were precipitated and cleaned in thestandard methods and resuspended into hexane for use in the shellreaction.

Shell Synthesis:

A 1:1 (w:v) mixture of tri-n-octylphosphine oxide (TOPO) andtri-n-octylphosphine (TOP) was introduced into a flask.Tetradecylphosphonic acid (TDPA) was added to the flask in an amountsuitable to fully passivate the final material, as can be calculatedfrom the reaction scale and the expected final nanoparticle size. Thecontents of the flask were heated to 125° C. under vacuum and then theflask was refilled with N₂ and cooled.

Inside the glovebox, a solution of a suitable cadmium precursor (such asdimethylcadmium or cadmium acetate) in TOP was prepared in a quantitysufficient to produce a desired thickness of shell, as can be calculatedby one of ordinary skill in the art. When a zinc shell was also desired,a solution of a suitable zinc precursor (such as diethylzinc or zincstearate) was prepared in TOP in a quantity sufficient to produce thedesired shell thickness. Separately, a solution of trimethylsilylsulfide[(TMS)₂S] in TOP was prepared in a quantity sufficient to produce thedesired shell thickness. Each of these solutions was taken up inseparate syringes and removed from the glove box.

Of the previously prepared core/hexane solution, 17 mL (at an opticaldensity of 21.5 at the band edge) was added to the reaction flask andthe hexane was removed by vacuum; the flask was then refilled with N2.The flask was heated to the desired synthesis temperature, typicallyabout 200 to about 250° C. During this heat-up, 17 mL of decylamine wasadded.

The cadmium and sulfur precursor solutions were then added alternatelyin layer additions, which were based upon the starting size of theunderlying cores. So this means that as each layer of shell material wasadded, a new “core” size was determined by taking the previous “core”size and adding to it the thickness of just-added shell material. Thisleads to a slightly larger volume of the following shell materialneeding to be added for each subsequent layer of shell material.

After a desired thickness of CdS shell material was added, the cadmiumprecursor solution was replaced with the zinc precursor solution. Zincand sulfur solutions were then added alternately in layer additionsuntil a desired thickness of ZnS was added. A final layer of the zincsolution was added at the end, the reaction flask was cooled, and theproduct was isolated by conventional precipitation methods.

Example 15: Exchange Process Using Dipeptide Ligands and Butanol as aCosolvent

Core/shell nanocrystals (quantum dots) were prepared by standardmethods, and were washed with acetic acid/toluene several times, andsuspended in hexanes. 10 nmol of core/shell nanocrystals were suspendedin 40 mL hexane. This was mixed with 10 mL of a 300 mM solution ofcarnosine and 10 mL of 1 M sodium carbonate solution. n-Butanol (14 mL)was added, and the vessel was flushed with argon. The mixture was mixedvigorously overnight at room temperature. The mixture was then heatedand allowed to cool to room temperature. The aqueous phase was thenremoved and filtered through a 0.2/0.8 micron syringe filter.

Excess carnosine was removed by dialyzing against 3.5 L of 25 mM NaClfor one hour. The solution was concentrated to 1 mL using a 10K MWCO(10,000 molecular weight cut-off) Amicon centricon. A solution was thenprepared with 568 mg of His-Leu dipeptide plus 212 mg of Gly-Hisdipeptide in 9 mL sodium carbonate solution, and this solution wascombined with the aqueous solution of quantum dots. This mixture wasstirred overnight at room temperature. The mixture of water-solublequantum dots was then dialyzed against 3.5 L of 25 mM NaCl for one hour.

To crosslink the peptide ligands (clarify) A solution of 0.5 mM4-aminobenzophenone in ethanol was then added to the aqueous quantumdots mixture, and the mixture was irradiated at 365 nm for 4 hours toeffect reaction of the aminobenzophenone with the surface molecules onthe quantum dots. To this, 5 mmol of THP (tris(hydroxymethyl)phosphine)was added, and the mixture was stirred at RT overnight, to inducecrosslinking. Another 5 mmol of THP was added, and again the mixture wasstirred overnight at RT. Another 5 mmol of THP was added the next day,along with 300 micromoles of PEG1000-COOH. This was mixed overnight atroom temperature, then another 5 mmol of THP was added along with 30mmol of glycine, and the mixture was stirred overnight at RT.

The material was purified by dialysis using the 10K MWCO Amiconcentricon, and was washed with 50 mM borate buffer (pH 9). The finalmaterial was dispersed into 50 mM borate buffer to a final concentrationof 2.5 micromolar for storage.

Example 16: Exchange Process Using Trithiol Ligands

A solution of hydrophobic phosphonate-coated quantum dots in organicsolvent (e.g. toluene, chloroform, etc) with a concentration of betweenabout 0.1 and 10 micromolar quantum dots was prepared. Approximately1000 to 1000000 equivalents of a suitable trithiol ligand was added,optionally as a solution in a suitable organic solvent (e.g. acetone,methanol, etc). The reaction mixture was stirred for 1-48 hours and thenthe solution was basicified by addition of an organic base (e.g.tetramethylammonium hydroxide, tetrabutylammonium hydroxide, etc). Aftera shorter second stirring period, water or aqueous buffer was used toextract the dots with hydrophilic ligands. The aqueous solution waswashed with additional organic solvent (e.g. toluene, chloroform, etc)and purified by filtration.

Example 17: Two-Step Ligand Exchange: Process for Exchanging PhosphonateLigands with Sulfonate (Triflate) Ligands

A nanoparticle comprising a core/shell nanocrystal having TDPA ligandson its surface is dissolved in dichloromethane, and excess TMS triflateis added to it. After 1-2 hours at room temperature, analysis indicatesthat the TDPA ligands have been removed, and the nanoparticle remainsdispersed in the solvent. It is dialyzed against dichloromethane using a10K MWCO (10,000 molecular-weight cut-off) dialysis membrane to removeexcess TMS triflate and the TMS-TDPA produced by the reaction of TMStriflate with the TDPA ligands. This produces a solution/suspension ofnanoparticles comprising triflate ligands on the surface ofnanocrystals. These triflate-containing nanoparticles are soluble inmany organic solvents, but may not be readily soluble in hexanes,depending upon the complement of ligands present.

Two-Step Process for Exchanging Sulfonate (Triflate) Ligands with PEGConjugated Dithiol (DHLA) Ligands Using n-Butanol as an IntermediateLigand and DMF as a Co-Solvent

The triflate-containing nanoparticle solution, described above, can becontacted with excess n-butanol in acetonitrile, using DMF as aco-solvent, to provide an intermediate nanoparticle believed to comprisebutanol ligands in place of the triflates which were on thenanoparticle. This intermediate nanoparticle can be isolated from themedium, or it can be further modified without isolation. Thisintermediate nanoparticle is contacted with an excess of a dihydrolipoicacid-PEG conjugate of this formula:

where n is 1-100.

The product is a water-soluble, stable nanoparticle. It can be collectedby extraction into a pH 9 buffer, and isolated by conventional methods,including dialysis with a 10K MWCO dialysis filter, or by size exclusion(gel filtration) chromatography.

Two-Step Process for Exchanging Sulfonate (Triflate) Ligands withNucleophilic Reactant Group Containing Ligands Using n-Butanol as anIntermediate Ligand and DMF as a Co-Solvent

The triflate-containing nanoparticle solution from can be contacted withexcess n-butanol in acetonitrile, using DMF as a co-solvent, to providean intermediate nanoparticle believed to comprise butanol ligands inplace of the triflates which were on the nanoparticle. This intermediatenanoparticle can be isolated from the medium, or it can be furthermodified without isolation. To further modify it, it is treated with anew ligand containing at least one nucleophilic reactant group: suitableligands include HS—CH₂—CH₂-PEG; aminomethyl phophonic acid;dihydrolipoic acid; omega-thio-alkanoic acids, andcarboxymethylphosphonic acid. The mixture is then treated with TMEDA(tetramethylethylene diamine), and monitored until triflate isdisplaced, then the nanocrystal product is extracted into pH 9 bufferand purified by conventional methods.

Process for Exchanging Sulfonate (Triflate) Ligands with CarboxylateFunctionalized Dithiol (DHLA) Ligands

The triflate-containing nanoparticle is contacted with neatdihydrolipoic acid (DHLA) for an hour at room temperature, and is thendispersed into pH 9 buffer and isolated by conventional methods. Thisprovides a nanoparticle having carboxylate groups to provide watersolubility, and having two thiol groups binding the carboxylate to thenanocrystal surface. The product is water soluble and stable in aqueousbuffer. It provides good colloidal stability, and a moderate quantumyield. This composition containing DHLA as a ligand contains freecarboxyl groups which can be used to attach other groups such as a PEGmoiety, optionally linked to a functional group or a biomolecule. Thesame reaction can be performed to replace triflate groups on ananoparticle with thioglycolic acid (HS—CH₂—COOH) ligands. This providesa highly stabilized nanoparticle which produces a high quantum yield,but has lower colloidal stability than the product having DHLA on itssurface.

Process for Exchanging Sulfonate (Triflate) Ligands with Amine Ligands

The triflate-containing nanoparticle is dispersed in dichloromethaneplus hexanes, and an alkylamine is added. Suitable alkylamines arepreferably primary amines, and include, e.g., H₂N—(CH₂)_(r)-PEG(r=2-10), p-aminomethylbenzoic acid, and lysine ethyl ester. After anhour at room temperature, the exchange process is completed, and thenanoparticle product can be isolated by conventional methods.

Process for Pre-Treating Phosphonate Coated Nanocrystals with TolueneAcetic Acid to Remove Impurities Prior to Exchanging with Sulfonate(Triflate) Ligands

TDPA-covered nanocrystals were synthesized which emitted light at 605 nmand had shells of CdS and of ZnS. These when treated with 200,000equivalents of TMS triflate in hexanes did not produce a precipitate.This was attributed to excess TDPA-derived impurities in thenanocrystals. This was alleviated by dissolving the nanocrystals intoluene-acetic acid and precipitating them with methanol, to remove TDPAsalts or related by-products. The resultant TDPA nanocrystals behaved asdescribed above, demonstrating that impurities were causing thenanocrystals to behave differently when made with excess TDPA present,and that those impurities can be removed by precipitation underconditions better suited to dissolving TDPA-related impurities.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals withDithiol: (DHLA) Ligands Using Butanol, DMF or Isopropyl Alcohol asDispersants

Three different methods of depositing the DHLA ligands were employed,each of which was considerably more rapid than the classic approachusing non-activated dots. In the first approach, the activated dotpowder was dispersed in butanol and stirred with DHLA, then precipitatedwith hexane and collected in aqueous buffer. In the second approach, theactivated dot powder was dispersed in dimethylformamide (DMF) andstirred with DHLA, then precipitated with toluene and collected inaqueous buffer. In the third approach, the activated dot powder wasstirred as a slurry in neat DHLA, then dispersed in isopropyl alcohol,precipitated with hexane, and collected in aqueous buffer and purifiedwith a filtration membrane.

These three samples, plus a sample derived from non-activated dots werediluted to 60 nM for a colloidal stability challenge, wherein theabsorbance is monitored over the course of days to watch forprecipitation. Samples 1 (butanol-mediated), 2 (DMF-mediated), and 4(classic) all precipitated on day 3 or 4 of the stability challenge, butsample 3 (neat DHLA) lasted twice as long, coming out of solution on day7. HPLC measurements indicated that the DHLA-coated particles producedfrom activated dots showed even less aggregation than the classic DHLAparticles made by the displacement of TOPO or pyridine ligands fromnanocrystals. Thus the invention provided rapid reactions leading toimproved colloidal stability and comparable or lower aggregation levelsthan conventional ligand replacement methods of putting DHLA on ananocrystal. Similar treatment with other thiol ligands likemercaptoundecanoic acid (MUA) or the PEGylated thiol also providedwater-dispersible nanocrystals. Reacting triflate-coated nanoparticleswith MUA or PEG-thiol gave particles which were readily dispersible inwater, indicating that ligand exchange had occurred. The observedquantum yield was over 70% in each case.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals withHydrophilic Phosphonate Ligands

Triflate-coated dots were dispersed in butanol and then stirred withphosphonoacetic acid. Triethylamine was added to form thetriethylammonium salt of both the phosphonate and carboxylatefunctionalities, and then pH 9 aqueous borate buffer was added toextract the hydrophilic particles. The result was a bright orangeaqueous dispersion of quantum dots, with no remaining color observed inthe butanol layer. The particles were purified by centrifugal filtrationand the quantum yield was measured to be 72%. Multiple batches ofparticles were prepared and remained in solution through roomtemperature storage for at least eight weeks. The same method can besuccessfully employed with DHLA, MUA, and PEGylated thiol ligands.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals with aVariety of Hydrophilic Phosphonate Ligands Via Biphasic Exchange

Using a biphasic exchange method, dispersing the quantum dots in organicsolvents such as chloroform and the exchangeable ligands in aqueoussolution, quantum dots were made water soluble and stable after ligandexchange with N,N-Bis(phosphonomethyl)glycine (1) or phosphonoaceticacid (2). In a typical bi-phasic ligand exchange experiment, 1 nmol ofquantum dots were dispersed in 1 mL of chloroform and placed in a vialwith 2 mL of 300 mM phosphonic acid in basic buffer and the mixture wasrapidly stirred at room temperature for 2 days. Quantum yields as highas 53% were achieved; however the quantum yields achieved were dependenton core-shell batch employed, probably as a result of variable amountsof long-chain alkyl phosphonates remaining on the nanocrystal surfacepost-ligand exchange. This demonstrated that complete removal of TDPAfrom nanocrystals is important for successful modification of thesurface. Though the dots were rendered water stable by the abovephosphonate-containing ligands, they were not successfully modified withPEG2000-diamine using standard EDC condensation chemistry.

Nanocrystals coated with compounds 1, 2, or 3 were readily prepared bythis method, as well as nanocrystals having a mixture of compounds 1 and2, or 1 and 3, or 2 and 3. In each case, the nanocrystals were stable,bright and water-soluble. Using mixed ligands, it was found thatPEGylation (with PEG2000-diamine using standard EDC condensationchemistry) could be achieved with these phosphonate-containing ligandsto produce highly stable, bright, water soluble nanoparticles. Thesenanoparticles can be further stabilized by at least partiallycross-linking the ligands using a diamine such as putrescine,cadaverine, 1,2-diaminoethane, bis(hexamethylene)triamine, PAMAMdendrimer, and cystamine.

Two-Step Ligand Exchange Process with Tridentate Thiol Ligands

Triflate exchange step was performed following the procedure describedabove. Next, the triflate nanoparticles were dispersed in organicsolvent (e.g. toluene, chloroform, etc) with a concentration of betweenabout 0.1 and 10 micromolar quantum dots. Approximately 1000 to 1000000equivalents of a suitable tridentate thiol ligand was added, optionallyas a solution in a suitable organic solvent (e.g. acetone, methanol,etc). The reaction mixture was stirred for 1-48 hours and then thesolution was basicified by addition of an organic base (e.g.tetramethylammonium hydroxide, tetrabutylammonium hydroxide, etc). Aftera shorter second stirring period, water or aqueous buffer was used toextract the dots with hydrophilic ligands. The aqueous solution waswashed with additional organic solvent (e.g. toluene, chloroform, etc)and purified by filtration.

Example 18

Functionalized Ligands on Nanoparticles

General Core Reaction Procedure

Into a 25 mL 3 neck flask with 14/20 joints, 1.575 g of >99%tri-n-octylphosphine oxide (TOPO) was weighed. To this, 1-1000micromoles of a bi-functional phosphonate ligand was added. A stir barwas added to this flask. The flask was connected to an inert atmospheremanifold and evacuated thoroughly, then refilled with nitrogen. Asolution of a suitable cadmium salt in tri-n-octylphosphine (TOP) wasprepared with a concentration of 0.5 mol Cd per kg solution. A desiredamount of cadmium as required for growth of nanoparticles of a desiredsize was extracted from this solution, diluted with 0.9 mL of additionalTOP, and added to the flask. The flask was stirred and heated to˜200-350° C. under nitrogen flow. A 1 molar solution of selenium in TOPwas prepared and a desired amount as required for growth ofnanoparticles of a desired size was added to the solution, optionallywith addition of a reaction promoter to achieve desired levels ofparticle nucleation. One minute after the reaction was initiated byadding these final reagents, a 20 microliter sample was removed from thereaction, mixed with 5 mL of hexane, and an emission spectrum wascollected. This aliquot removal and measurement process was repeatedafter 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 minutes. After 14 minutes, thereaction was rapidly cooled and the products were isolated by methodsunderstood in the art.

Control Core Reaction with Tetradecylphosphonic Acid [TDPA]

The core reaction using TDPA as the phosphonate ligand was demonstratedas a control reaction. This reaction proceeded with an initial emissionreading at 1 minute of ˜490 nm and progressing to a final emissionreading of ˜544 nm at 14 minutes. The full width at half maximumintensity (FWHM) never got above 28 nm. The final “growth solution” ofthe cores was yellow/light orange in appearance by eye. The aliquotedsamples of this reaction remained dispersed and clear solutions inhexane.

Core Reaction with 11-methoxy-11-oxo-undecylphosphonic acid

The reaction using 11-methoxy-11-oxo-undecylphosphonic acid as thephosphonate ligand proceeded with an initial emission reading at 1minute was ˜560 nm; this was redder than the final emission of thecontrol reaction. The final emission of this reaction was ˜610 nm. TheFWHM of this reaction started at ˜35 nm and steadily got more broadthroughout the reaction for a final FWHM of ˜50 nm.

The aliquoted samples were not soluble in hexane, and became almostinstantly flocculated and settled to the bottom of the vials withinminutes.

Core Reaction with 6-ethoxy-6-oxohexylphosphonic acid

The core reaction using 6-ethoxy-6-oxohexylphosphonic acid as thephosphonate ligand had an initial emission reading at 1 minute of ˜560nm and a final emission reading of ˜606 nm. The FWHM of this reactionstarted out at 1 minute at ˜43 nm and narrowed to a final FWHM of ˜40.5nm.

The solubility of the aliquoted samples was observed. The hexane sampleswere immediately cloudy, however the flocculation did not settle to thebottom of the vials. Six of the aliquoted samples were centrifuged andthe resulting clear, colorless supernatants were discarded. The pelletswere soluble in toluene, dichloromethane (CH₂Cl₂), dimethylformamide(DMF), and methanol (MeOH). The pellets were not soluble in water, 50 mMborate buffer at pH=8.3 or hexane.

Particles synthesized in the presence of TDPA are soluble in hexane,toluene, CH₂Cl₂, DMF and hexane. The 6-ethoxy-6-oxohexylphosphonic aciditself is not soluble in hexane, and neither were the resultingparticles from this reaction, suggesting that the ligand was indeedcoating the nanoparticles—a suggestion which was confirmed with infraredand NMR spectroscopy indicating the expected ester functionality. Usinga solvent system of toluene as the solubilizing solvent and hexane as aprecipitating solvent, a pellet can be formed along with a clear,colorless supernatant. The resulting pellet can be re-solubilized intoluene. This resulting toluene solution allowed an absorbance spectrumof these cores to be obtained.

These data suggest that quantum confined nanoparticles have been formedwith 6-ethoxy-6-oxohexylphosphonic acid on the particle surface. Theresulting core particles were taken further into a shell reaction.

Shell Reaction Procedure Using 6-ethoxy-6-oxohexylphosphonic acid

Core Precipitation

Three (3) mL of growth solution cores using6-ethoxy-6-oxohexylphosphonic acid ligand (prepared according to theprocedure of Example 17) was solubilized into 3 mL toluene in a 250 mLconical bottom centrifuge tube. A total of 135 mL of hexane was added toprecipitate the cores. The tube was centrifuged at 3000 RPM for 5 min.The resulting clear, colorless supernatant was discarded and the pelletwas dispersed into 3 mL of toluene.

Shell Reaction

Into a 25 mL 3 neck flask with 14/20 joints, 1.4 g of TOPO was weighed.To this, 1-1000 mg of 6-ethoxy-6-oxohexylphosphonic acid was added. Astir bar and 1.4 mL of TOP were added to the flask. The flask wasconnected to an inert atmosphere manifold and evacuated thoroughly, thenrefilled with nitrogen. 2.6 mL of the toluene solution of cores wasadded to the flask and the flask was warmed and evacuated to remove thetoluene, then refilled with nitrogen. Approximately 1 mL of a suitablyhigh-boiling amine was added to the flask and the flask was heated to200-350° C. Solutions of suitable cadmium and zinc precursors in TOPwere prepared with a concentration of 0.5 mol metal ion per kg ofsolution. A solution of 10% trimethylsilylsulfide in TOP by weight wasprepared as well. The metal and sulfur precursor solutions were addedslowly over the course of several hours to minimize additionalnanoparticle nucleation. Sufficient shell precursors were added to growa shell of a desired thickness, as can be calculated by one of ordinaryskill in the art. When the desired shell thickness was reached, thereaction was cooled and the core/shell nanoparticles were isolated byconventional means. Aliquots taken during the reaction permittedmonitoring of the progress of the shell reaction. It was observed thatthe emission maximum after heating but before addition of shellprecursors was very similar to that of the initial cores (˜600 nm),suggesting that the bi-functional phosphonate was sufficiently stronglycoordinated to the nanoparticle surface to minimize Ostwald ripening. Ared-shift during shell precursor addition of ˜50 nm was typical of ashell as deposited in a reaction employing TDPA, suggesting that theshell formed as expected. In addition, the nanoparticle solution becamemuch more intensely emissive, as would be expected of successfuldeposition of an insulating shell. Infrared and NMR spectroscopyconfirmed that the functionalized phosphonates were present on thenanoparticles.

What is claimed is:
 1. A method for performing a primer extensionreaction, comprising: contacting a modified polymerase comprising anamino acid sequence that is at least 98% identical to the amino acidsequence of SEQ ID NO: 7 with a nucleic acid molecule and at least onenucleotide under conditions where the at least one nucleotide isincorporated into the nucleic acid molecule by the polymerase; whereinthe modified polymerase has amino acid leucine at position 383 and aminoacid asparagine at position 384 and one or more additional amino acidmodifications, and the modified polymerase exhibits increased polymeraseactivity relative to the unmodified reference B103 polymerase, SEQ IDNO: 6, and wherein the one or more additional amino acid modificationsof the modified polymerase consist of: i) one or more amino acidsubstitutions, additions or deletions at one or more positions selectedfrom the group consisting of: 9, 11, 12, 58, 59, 63, 162, 166, 377 and385, and the modified polymerase exhibits reduced exonuclease activityrelative to an unmodified B103 polymerase, SEQ ID NO: 6; ii) one or moreamino acid substitutions at one or more positions selected from thegroup consisting of: 365, 370, 371, 372, 481, 507, and 509, wherein theone or more amino acid substitutions at position 372 is selected fromthe group consisting of K372G, K372T, K372S, K372R, K372A, K372Q, K372W,K372Y, and K372F, and the modified polymerase exhibits an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference modified polymerase of SEQ ID NO: 7; and/or iii) an aminoacid substitution at position 380, and the modified polymerase exhibitsincreased t_(po1) value (or decreased k_(po1) value) relative to theunmodified B103 polymerase, SEQ ID NO:
 6. 2. The method of claim 1,wherein the at least one nucleotide is a labeled nucleotide, and thelabel of the nucleotide emits a signal during incorporation of thenucleotide.
 3. The method of claim 2, further comprising detecting thesignal emitted by the nucleotide label.
 4. The method of claim 2,wherein the labeled nucleotide is a reversible terminator.
 5. The methodof claim 2, wherein the modified polymerase is linked to a label.
 6. Amethod for detecting one or more nucleotide incorporations onto the endof a single nucleic acid molecule, comprising: contacting a targetnucleic acid molecule with a modified polymerase comprising an aminoacid sequence that is at least 98% identical to the amino acid sequenceof SEQ ID NO: 7 with one or more labeled nucleotides under conditionswhere at least one labeled nucleotide is incorporated by the polymeraseonto the end of an extending nucleic acid molecule, wherein the label ofat least one labeled nucleotide emits a signal indicative of nucleotideincorporation; and detecting the one or more signals indicative ofnucleotide incorporation, wherein the modified polymerase has amino acidleucine at position 383 and amino acid asparagine at position 384 andone or more additional amino acid modifications, and the modifiedpolymerase exhibits increased polymerase activity relative to theunmodified reference B103 polymerase, SEQ ID NO: 6, and wherein the oneor more additional amino acid modifications of the modified polymeraseconsist of: i) one or more amino acid substitutions, additions ordeletions at one or more positions selected from the group consistingof: 9, 11, 12, 58, 59, 63, 162, 166, 377 and 385 and the modifiedpolymerase exhibits reduced exonuclease activity relative to anunmodified B103 polymerase, SEQ ID NO: 6; ii) one or more amino acidsubstitutions at one or more positions selected from the groupconsisting of: 365, 370, 371, 372, 481, 507, and 509, wherein the one ormore amino acid substitutions at position 372 is selected from the groupconsisting of K372G, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y,and K372F, and the modified polymerase exhibits an increased branchingratio and/or increased nucleotide binding affinity relative to areference modified polymerase of SEQ ID NO: 7; and/or iii) an amino acidsubstitution at position 380 and the modified polymerase exhibitsincreased t_(po1) value (or decreased k_(po1) value) relative to theunmodified B103 polymerase, SEQ ID NO:
 6. 7. The method of claim 6,further comprising analyzing the one or more detected signals emitted bythe nucleotide label to determine the presence of a target nucleic acidmolecule.
 8. The method of claim 6 wherein the detecting is performed inreal time or near real time of the incorporation.
 9. The method of claim6, wherein the labeled nucleotide is a reversible terminator.
 10. Themethod of claim 6, wherein the modified polymerase is linked to a label.11. The method of claim 7 comprising identifying the target nucleic acidmolecule.
 12. The method of claim 7 comprising determining the sequenceof some or all of the sequence of the target nucleic acid molecule. 13.A method for sequencing a nucleic acid molecule, comprising: (a)conducting a polymerase reaction comprising contacting a target nucleicacid molecule with a modified polymerase comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 7 with one or more labeled nucleotides under conditions whereat least one labeled nucleotide is incorporated by the polymerase ontothe end of an extending nucleic acid molecule, wherein the targetnucleic acid molecule is paired with a polymerization initiation siteand wherein the label of at least one labeled nucleotide emits a signalindicative of nucleotide incorporation; and (b) detecting a timesequence of nucleotide incorporations from the one or more signalsindicative of nucleotide incorporation; and (c) determining the identityof one or more incorporated nucleotides, thereby determining some or allof the nucleotide sequence of a single nucleic acid molecule, whereinthe modified polymerase has amino acid leucine at position 383 and aminoacid asparagine at position 384 and one or more additional amino acidmodifications, and the modified polymerase exhibits increased polymeraseactivity relative to the unmodified reference B103 polymerase, SEQ IDNO: 6, and wherein the one or more additional amino acid modificationsof the modified polymerase consist of: i) one or more amino acidsubstitutions, additions or deletions at one or more positions selectedfrom the group consisting of: 2-9, 11, 12, 58, 59, 63, 162, 166, 377 and385 and the modified polymerase exhibits reduced exonuclease activityrelative to an unmodified B103 polymerase, SEQ ID NO: 6; ii) one or moreamino acid substitutions at one or more positions selected from thegroup consisting of: 365, 370, 371, 372, 481, 507, and 509, wherein theone or more amino acid substitutions at position 372 is selected fromthe group consisting of K372G, K372T, K372S, K372R, K372A, K372Q, K372W,K372Y, and K372F, and the modified polymerase exhibits an increasedbranching ratio and/or increased nucleotide binding affinity relative toa reference modified polymerase of SEQ ID NO: 7; and/or iii) an aminoacid substitution at position 380 and the modified polymerase exhibitsincreased t_(po1) value (or decreased k_(po1) value) relative to theunmodified B103 polymerase, SEQ ID NO:
 6. 14. The method of claim 13,further comprising analyzing each of the one or more detected signalsemitted by the nucleotide label to determine the sequence of a targetnucleic acid molecule.
 15. The method of claim 13 wherein the detectingis performed in real time or near real time of the incorporation. 16.The method of claim 13, wherein, the labeled nucleotide comprises anucleotide label linked to the base, sugar, or phosphate group of thenucleotide.
 17. The method of claim 13, wherein the labeled nucleotideis a reversible terminator.
 18. The method of claim 13, wherein themodified polymerase is linked to a label.
 19. The method of claim 17wherein the reaction is detected and monitored in real time based ondetection of FRET signals.
 20. The method of claim 14 comprisingdetermining the sequence of some or all of the sequence of the targetnucleic acid molecule.